Smooth muscle specific inhibition for anti-restenotic therapy

ABSTRACT

The present invention provides for the incorporation of target sequences of microRNAs into the 3′ UTR region of a gene of interest in nucleic acid vectors. The invention also provides for an expression system comprising such vectors, a pharmaceutical composition comprising such vectors, as well as methods of treating or preventing cardiovascular disease by using such vectors.

This application claims priority to U.S. Provisional Application No.61/620,404, filed Apr. 4, 2012 and U.S. Provisional Application No.61/727,003, filed Nov. 15, 2012, the contents of which are incorporatedby reference herein in their entireties.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No.K99HL109133-01 awarded by the National Institute of Health. TheGovernment has certain rights in the invention.

This patent disclosure contains material that is subject to copyrightprotection. The copyright owner has no objection to the facsimilereproduction by anyone of the patent document or the patent disclosureas it appears in the U.S. Patent and Trademark Office patent file orrecords, but otherwise reserves any and all copyright rights.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosure ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described herein.

BACKGROUND OF THE INVENTION

The high incidence of cardiovascular diseases represents a significantmedical problem in the USA and throughout the world, despite remarkableadvances over the past few decades. From the 1990's to early 2000's,restenosis after balloon angioplasty and/or stent implantation occurredwith an incidence of approximately 50% and 25%, respectively, leading toa significant limitation in its effectiveness.

A major advance in the treatment of coronary artery disease (CAD)emerged from studies showing that rapamycin was a potent inhibitor ofproliferation and migration of VSMCs, and led to the development ofrapamycin-coated stents (Cypher) which markedly reduced restenosis andrevolutionized the field of percutaneous coronary intervention (PCI).

Percutaneous coronary intervention (PCI) is one of the most commonlyperformed interventions that have transformed the practice ofrevascularization for CAD. However, the major drawback of this procedureis the proliferation and subsequent accumulation of vascular smoothmuscle cells (VSMC), leading to restenosis. The advent of drug elutingstents, capable of delivering an inhibitor of cell proliferation in situhas decreased, but not eliminated, the occurrence of restenosis. Thedrugs that elute from the stent not only inhibit VSMC, but also ECproliferation and migration, increasing the risk of late thrombosis.Thus, there is a need for therapies that would provide VSMC selectiveanti-proliferative activity without affecting EC.

SUMMARY OF THE INVENTION

The present invention provides for the incorporation of target sequencesfor a microRNA into the 3′-UTR of a gene, such as, but not limited to, atumor suppressor gene, that is present in an expression vector. Theexpression vector is used to specifically over-express the gene in onecell type that either does not express the microRNA, or has very lowexpression levels of the microRNA, while inhibiting the expression ofthe gene in another cell type that has higher expression levels of themicroRNA.

In one aspect, the present invention provides a nucleic acid vectorcomprising a gene of interest and one or more target sequences for amicroRNA within the 3′ UTR region of the gene of interest. In oneembodiment, the gene of interest is the p27 gene. In one embodiment, themicroRNA is miR-126. In one embodiment, the one or more target sequencescomprise SEQ ID NO:2. In one embodiment, the vector comprises fourtarget sequences for a microRNA. In another embodiment, the targetsequences for a microRNA are identical. In one embodiment, the targetsequences for a microRNA comprise SEQ ID NO:2.

In another embodiment, the vector comprises a second gene of interest.In another embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.

In another embodiment, the nucleic acid vector is a viral vector. In yetanother embodiment, the viral vector is an adenoviral vector. In anotherembodiment, the vector is delivered to a cell of interest. In oneembodiment, the cell of interest is an endothelial cell.

In another aspect, the invention provides an expression systemcomprising a nucleic acid vector, wherein the nucleic acid vectorcomprises a gene of interest, and one or more target sequences for amicroRNA within the 3′ UTR region of the gene of interest. In oneembodiment, the gene of interest is the p27 gene. In one embodiment, themicroRNA is miR-126. In one embodiment, the one or more target sequencescomprise SEQ ID NO:2. In one embodiment, the vector comprises fourtarget sequences for a microRNA. In another embodiment, the targetsequences for a microRNA are identical. In one embodiment, the targetsequences for a microRNA comprise SEQ ID NO:2.

In another embodiment, the vector comprises a second gene of interest.In another embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In another embodiment, the nucleic acid vector is a viral vector. In yetanother embodiment, the viral vector is an adenoviral vector.

In another aspect, the invention provides a cell comprising a nucleicacid vector, wherein the nucleic acid vector comprises a gene ofinterest, and one or more target sequences for a microRNA within the 3′UTR region of the gene of interest. In one embodiment, the gene ofinterest is the p27 gene. In one embodiment, the microRNA is miR-126. Inone embodiment, the one or more target sequences comprise SEQ ID NO:2.In one embodiment, the vector comprises four target sequences for amicroRNA. In another embodiment, the target sequences for a microRNA areidentical. In one embodiment, the target sequences for a microRNAcomprise SEQ ID NO:2.

In one embodiment, the vector comprises a second gene of interest. Inanother embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In one embodiment, the nucleic acid vector is a viral vector. In yetanother embodiment, the viral vector is an adenoviral vector. In oneembodiment, the cell is an endothelial cell. In another embodiment, thecell is a vascular smooth muscle cell.

In one aspect, the invention provides a pharmaceutical compositioncomprising a nucleic acid vector, wherein the nucleic acid vectorcomprises a gene of interest, and one or more target sequences for amicroRNA within the 3′ UTR region of the gene of interest. In oneembodiment, the gene of interest is the p27 gene. In one embodiment, themicroRNA is miR-126. In one embodiment, the one or more target sequencescomprise SEQ ID NO:2. In one embodiment, the vector comprises fourtarget sequences for a microRNA. In another embodiment, the targetsequences for a microRNA are identical. In one embodiment, the targetsequences for a microRNA comprise SEQ ID NO:2.

In one embodiment, the vector comprises a second gene of interest. Inanother embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In another embodiment, the nucleic acid vector is a viral vector. In yetanother embodiment, the viral vector is an adenoviral vector.

In another aspect, the invention provides a method of treating orpreventing a cardiovascular disease in a subject in need thereof, themethod comprising administering a nucleic acid vector to the subject,wherein the nucleic acid vector comprises a gene of interest and one ormore target sequences for a microRNA within the 3′ UTR region of thegene of interest. In one embodiment, the gene of interest is the p27gene. In one embodiment, the microRNA is miR-126. In one embodiment, theone or more target sequences comprise SEQ ID NO:2. In one embodiment,the vector comprises four target sequences for a microRNA. In anotherembodiment, the target sequences for a microRNA are identical. In oneembodiment, the target sequences for a microRNA comprise SEQ ID NO:2.

In one embodiment, the vector comprises a second gene of interest. Inanother embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In another embodiment, the nucleic acid vector is a viral vector. In yetanother embodiment, the viral vector is an adenoviral vector.

In one embodiment, the nucleic acid vector is delivered into a cell ofthe subject. In another embodiment, the cell expresses the microRNA. Inone embodiment, the gene of interest is not expressed in the cell. Inone embodiment, the cell is an endothelial cell. In another embodiment,the cell is a vascular smooth muscle cell. In one embodiment, thecardiovascular disease is coronary artery disease.

These, and other, embodiments of the invention will be betterappreciated and understood when considered in conjunction with thefollowing description and the accompanying drawings. It should beunderstood, however, that the following description, while indicatingvarious embodiments of the invention and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions and/or rearrangements may bemade within the scope of the invention without departing from the spiritthereof, and the invention includes all such substitutions,modifications, additions and/or rearrangements.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-D. Schematic representation of p27-expressing viruses. FIG. 1A.Control CMV virus (CMV) FIG. 1B. p27 expressing adenovirus under thecontrol of CMV promoter (p27). FIG. 1C. p27 expressing adenovirus underthe control of CMV promoter, containing four target sequences formir-126 in their 3′UTR (p27-126TS). FIG. 1D. Representative immunoblotsfor the indicated proteins from VSMC or HUVEC infected with adenovirusdescribed in A-C at 20 pfu/cell or 50 pfu/cell respectively for 48 hr.

FIGS. 2A-J. Ad-p27-126TS preserves EC function in vitro. FIG. 2A. RealTime qRT-PCR analysis of miR-126 (FIGS. 2B-C) Representative immunoblotof three independent experiments quantified by densitometry. FIG. 2D.Proliferation assays of VSMC and EC infected with the indicated Ad.Migration assays of VSMC (FIGS. 2E, F) and EC (FIGS. 2G, H) infectedwith the indicated Ad; representative pictures at the indicated timepoints are shown. FIGS. 2I, J. Network formation assay in EC. Orangescale bar indicates 100 μm (magnification 4×). All data shown aremeans±s.e.m. from at least three experiments performed in quadruplets.Data comparisons were made using one way analysis of variance withBonferroni post hoc tests; *P<0.01 versus VSMC (FIG. 2A); *P<0.01 versusAd-GFP (FIGS. 2C, D, F, H, J).

FIGS. 3A-E. Effects of Ad-p27-126TS in an in vivo model of arterialinjury. FIG. 3A. Representative sections of rat carotid arteriesimmunostained for smooth muscle cell actin (αSMA) and for the specificEC marker VE-Cadherin (VE-Cad). Nuclei were counterstained with DAPI. Nopositive staining was observed in the negative control sections. Orangescale bars: 100 μm (magnification 60×, inlays show high magnificationimages), arrowheads indicate EC beyond the inner autofluorescent elasticlaminae. FIG. 3B. Neointima/media ratios and (FIG. 3C) endothelialcoverage were calculated from 5 rats/group. FIG. 3D. Plasma levels ofD-dimer in plasma collected before (uninjured) and 2 weeks after theballoon injury using a specific rat immunoassay; n=4 rats/group. FIG.3E. Vascular reactivity analysis on carotid rings showing thevasodilatative response to acetylcholine (ACh); n=3 rats/group. All thedata are means±s.e.m. from at least three experiments performed intriplicate. Data comparison was made using one way (FIGS. 3B-D) ortwo-way repeated measures (FIG. 3E) analysis of variance with Bonferronipost hoc tests; *P<0.01 versus uninjured.

FIGS. 4A-C. Schematic representation of the used adenoviruses (Ad). ApAdTrack-CMV vector that contains a green fluorescence protein (GFP)under the control of a separate CMV promoter was used to generate threeadenoviruses: FIG. 4A. The Ad-GFP contains only the sequence for GFP.FIG. 4B. The Ad-p27 contains the sequences for GFP and p27. FIG. 4C. TheAd-p27-126TS contains GFP and p27 that have four target sequences formiR-126 in its 3′UTR.

FIGS. 5A-B. Histological assessment of rat carotid arteries 2 weeksafter injury and adenoviral infection. FIG. 5A. To overcome theautofluorescence issues typical of the vascular sections, the efficiencyof the Ad infection was evaluated by using a primary antibody againstGFP, revealed by a Cy3-conjugated secondary antibody. Representativedigital images are shown. Orange scale bar represents 100 μm(magnification 60×). FIG. 5B. Representative sections stained withhematoxilyn/eosin (magnification 10×, inlays show the whole arterialsection in a 5× magnification; orange scale bar represents 500 μm).

DETAILED DESCRIPTION OF THE INVENTION

The patent and scientific literature referred to herein establishesknowledge that is available to those skilled in the art. The issuedpatents, applications, and other publications that are cited herein arehereby incorporated by reference to the same extent as if each wasspecifically and individually indicated to be incorporated by reference.

As would be apparent to one of ordinary skill in the art, any method orcomposition described herein can be implemented with respect to anyother method or composition described herein.

ABBREVIATIONS AND DEFINITIONS

As used herein, the term “p27” refers to the cyclin-dependent kinaseinhibitor also known as p27^(Kip1), which is a member of the kinaseinhibitor protein (KIP) family. p27 is a protein encoded by the CDKN1Bgene. The nucleic acid sequences of the gene encoding p27, including,but not limited to, the nucleic acid sequence of the open reading frameof the gene, is known in the art. The nucleic acid sequences of the geneencoding human p27, including, but not limited to, the nucleic acidsequence of the open reading frame of the human gene, is known in theart. The amino acid sequences of the p27 polypeptide and protein,including, but not limited to, the amino acid sequences of the human p27polypeptide and proteins, are known in the art. The GeneBank accessionnumber of the nucleic acid sequence of human p27 is NM_(—)004064. TheGeneBank accession number of the amino acid sequence of human p27 isNP_(—)004055. For additional information on p27, see e.g., Nisar P.Malek, Holly Sundberg, Seth McGrew, Keiko Nakayama, Themis R. Kyriakidis& James M. Roberts, 2001, A mouse knock-in model exposes sequentialproteolytic pathways that regulate p27Kip1 in G1 and S phase, Nature,413, 323-327. p27 is also known as KIP1; MEN4; CDKN4, MEN1B and P27KIP1.

As used herein, the term “miR-126” refers to microRNA 126. The nucleicacid sequence of miR-126 is known in the art. The nucleic acid sequencesof human miR-126 is known in the art. The GeneBank accession number ofthe nucleic acid sequence of human miR-126 is M10000471.

As used herein, the abbreviation “HUVEC” refers to Human UmbilicalVenous Endothelial Cells.

As used herein, the abbreviation “EC” refers to Endothelial Cells.

As used herein, the abbreviation “VSMC” refers to Vascular Smooth MuscleCells.

As used herein, the abbreviation “SMC” refers to smooth muscle cells.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one.”and “one or more than one.”

The term “about” is used herein to mean approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20%.

DETAILED DESCRIPTION

Treatment of Coronary Artery Disease

Cardiovascular disease accounts for nearly one third of deaths globally,and Coronary Artery Disease (CAD) remains the number one cause of deathin the United States (Heron M, Hoyert D L, Murphy S L, Xu J, Kochanek KD, Tejada-Vera B: Deaths: final data for 2006. Natl Vital Stat Rep57(14), 1-134 (2009)).

The introduction of the Drug-Eluting Stent (DES) has revolutionized thefield of angioplasty, by significantly reducing rates of restenosis whencompared to bare-metal stents (BES) (Marks A R: Sirolimus for theprevention of in-stent restenosis in a coronary artery. N Engl J Med349(14), 1307-1309 (2003). The anti-restenotic action of the drug(Sirolimus or rapamycin) lies at least in part, on its ability toup-regulate levels of p27, a widely expressed protein that inhibitscyclin-dependent kinases (cdks) complexes in G1 and S phases (Marx S O,Totary-Jain H, Marks A R: Vascular smooth muscle cell proliferation inrestenosis. Circ Cardiovasc Intenr 4(1), 104-111 (2011). Despite thesignificant improvement, drug-eluting stents (DES) hike the risk of latestent thrombosis by about 0.2% over bare-metal stents and is associatedwith long-term myocardial infarction and death rates (Garg S, Serruys PW: Coronary stents: looking forward. J Am Coll Cardiol 56(10 Suppl).S43-78 (2010): Wijns W: Late stent thrombosis after drug-eluting stent:seeing is understanding. Circulation 120(5), 364-365 (2009)). Delayedendothelial coverage after DES implantation is thought to prolong thewindow of vulnerability to stent thrombosis, which requires a prolongeddual anti-platelet therapy (Van Den Heuvel M, Sorop O, Van Beusekom H M,Van Der Giessen W J: Endothelial dysfunction after drug eluting stentimplantation. Minerva Cardioangiol 57(5), 629-643 (2009)).

The current generation of DES that elute non selective drugs from theirsurfaces to reduce smooth muscle cell growth also inhibitendothelialisation (Finn A V, Nakazawa G, Joner M el al.: Vascularresponses to drug eluting stents: importance of delayed healing.Arterioscler Thromb Vasc Biol 27(7), 1500-1510 (2007); Joner M, Finn AV, Farb A et al.: Pathology of drug-eluting stents in humans: delayedhealing and late thrombotic risk. J Am Coll Cardiol 48(1), 193-202(2006)).

Newer stents and coronary devices are currently undergoing pre-clinicaland clinical trials including DES with biodegradable polymers, polymerfree DES, new coated stents, completely biodegradable stents,bifurcation stents and drug-eluting balloons. However, these newapproaches continue to utilize non-selective antiproliferative drugsthat inhibit both vascular smooth muscle cells (VSMC) and vascularendothelial cell (EC) proliferation, adversely affecting criticalendothelial cell functions such as maintaining vascular tone, providinga permeable barrier, modulating adhesion, inflammation and thrombosis.Therefore, it is of paramount importance to develop therapeuticstrategies that can selectively inhibit VSMC and other infiltrated cellswithout affecting EC functions. The present invention addresses thisneed.

The present invention provides for the incorporation of target sequencesfor a microRNA into the 3′-UTR of a gene, such as, but not limited to, atumor suppressor gene, that is present in an expression vector. Theexpression vector is used to specifically over-express the gene in onecell type that either does not express the microRNA, or has very lowexpression levels of the microRNA, while inhibiting the expression ofthe gene in another cell type that has higher expression levels of themicroRNA.

In one embodiment, the present invention provides for the incorporationof target sequences for the VEC-specific miRNA, miR-126, into the 3′-UTRof p27 in a p27-expressing vector, to specifically over-express p27 invascular smooth muscle cells (VSMC), without affecting endothelial cells(EC) that express mir-126 at higher levels than VSMC. This strategy willresult in the specific inhibition of VSMC proliferation, migration andneointimal formation, without affecting endothelial cells, becausep27-expressing vectors will be targeted for degradation in endothelialcells.

The invention can be used, for example, in conjunction with BMSimplantation to provide the benefits of a DES without the concern of DESpost-operative complications. The invention can be used, for example,for the inhibition of VSMC proliferation in altherosclerosis andarterial injury, as well as vascular access failure in hemodialysispatients.

MicroRNAs

A variety of nucleic acid species are capable of modifying geneexpression. These include antisense RNA, siRNA, microRNA, RNA and DNAaptamers, and decoy RNAs. Each of these nucleic acid species can inhibittarget nucleic acid activity, including gene expression.

MicroRNAs (miRNAs or miRs) are a class of short (18-25 nt) non-codingRNAs (ncRNAs) that exist in a variety of organisms, including mammals,and are conserved in evolution. miRNAs are processed from hairpinprecursors of 70 nt (pre-miRNA) which are derived from primarytranscripts (pri-miRNA) through sequential cleavage by the RNAse IIIenzymes drosha and dicer. miRNAs can be encoded in intergenic regions,hosted within introns of pre-mRNAs or within ncRNA genes. Many miRNAsalso tend to be clustered and transcribed as polycistrons and often havesimilar spatial temporal expression patterns. miRNAs have been found tohave roles in a variety of biological processes including developmentaltiming, differentiation, apoptosis, cell proliferation, organdevelopment, and metabolism.

At least 222 separate miRNA genes have been identified in the humangenome. For example, 2 miRNA genes (miR15a and miR16a) have beenlocalized to a homozygously deleted region on chromosome 13 that iscorrelated with chronic lymphocytic leukemia (Calin et al. (2002), Proc.Natl. Acad. Sci. USA 99:15524-29). However, the distribution of miRNAgenes throughout the genome, and the relationship of the miRNA genes todiverse chromosomal features, has not been systematically studied. Afurther review of miRNAs is provided in U.S. Pat. No. 7,232,806, U.S.Patent Application Publication No. 2006/0105360, and in the references:Landgraf et al., 2007, Cell 129: 1401-1414; Mendell, J T, 2005 CellCycle 4(9):1179-84; Shivdasani R A, 2006 Blood 108(12):3646-53; Hwangand Mendell, 2006 Br J Cancer 94(6):776-80; Hammond S M, 2006: Curr OpinGenet Dev. 16(1):4-9; Osada and Takahashi, 2007 Carcinogenesis28(1):2-12; and Zhang et al., 2007 Dev Biol. 302(1):1-12, all of whichare hereby incorporated by reference in their entirety.

MicroRNAs can inhibit target nucleic acid activity, including geneexpression. For example, miRNAs can function via base paring withcomplementary nucleic acid sequences within mRNA molecules. The pairingof miRNAs with complementary mRNA molecules usually results in genesilencing via translational repression or target degradation. AnimalmiRNAs can exhibit only partial complementarity to their mRNA targets. Aseed region of about 6-8 nucleotides in length at the 5′ end of an miRNAis thought to be an important determinant of target specificity. A givenmiRNA can have multiple different mRNA targets and a given target can betargeted by multiple miRNAs. For additional information on miRNAs, seealso Ishida el al., 2013, miRNA-Based Therapeutic Strategies, CurrAnesthesiol Rep., 1(1):63-70, which is incorporated herein by referencein its entirety.

Nucleic Acid Vectors and Expression Systems

In one aspect, the present invention provides a nucleic acid vectorcomprising a gene of interest, and one or more target sequences for amicroRNA within the 3′ UTR region of the gene of interest.

In one embodiment, the gene of interest is a human gene. In anotherembodiment, the gene of interest is a non-human gene. In one embodiment,the gene of interest is a p27 gene. In another embodiment, the gene ofinterest is p53. In other embodiments, the gene of interest is a tumorsuppressor gene. A tumor suppressor gene can include, but is not limitedto, APC, RB1, INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4.

In one embodiment, the microRNA is miR-126. In another embodiment, themicro-RNA is miR-143. In another embodiment, the micro-RNA is miR-145.In further embodiments, the microRNA is Let7-f, miR-27b, miR-130a,miR-221, miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a,miR-378, miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the one or more target sequences comprise SEQ IDNO:2. In another embodiment, the one or more target sequences do notcomprise SEQ ID NO:2. In another embodiment, the one or more targetsequences comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the one or more target sequences do not comprisea nucleic acid sequence complementary to SEQ ID NO:2.

In one embodiment, the vector comprises four target sequences for amicroRNA. In another embodiment, the vector comprises three targetsequences. In another embodiment, the vector comprises four targetsequences. In another embodiment, the vector comprises five targetsequences. In another embodiment, the vector comprises six targetsequences. In another embodiment, the vector comprises seven targetsequences. In another embodiment, the vector comprises eight targetsequences. In other embodiments, the vector comprises nine, ten, eleven,twelve, thirteen, fourteen, fifteen, or more target sequences.

In one embodiment, the target sequences for a microRNA are identical. Inanother embodiment, the target sequences for a microRNA are notidentical. The nucleic acid vectors can comprise different combinationsof target sequences for various microRNAs, including, but not limitedto, miR-126, miR-143, miR-145, Let7-f, miR-27b, miR-130a, miR-221,miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a, miR-378,miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the target sequences for a microRNA comprise SEQ IDNO:2. In another embodiment, the target sequences for a microRNA do notcomprise SEQ ID NO:2. In another embodiment, the target sequences for amicroRNA comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the target sequences for a microRNA do notcomprise a nucleic acid sequence complementary to SEQ ID NO:2.

In another embodiment, the vector comprises a second gene of interest.In one embodiment, the second gene of interest is a human gene. Inanother embodiment, the second gene of interest is a non-human gene. Inone embodiment, the second gene of interest is p53. In otherembodiments, the second gene of interest is a tumor suppressor gene. Atumor suppressor gene can include, but is not limited to, APC, RB1,INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4. In one embodiment, the secondgene of interest is an antithrombotic gene. In another embodiment, thesecond gene of interest is an anti-inflammatory gene. In anotherembodiment, the second gene of interest is ENTPD1, TFPI or PTGIS.

In another embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In one embodiment, the vector comprises one target sequence. In anotherembodiment, the vector comprises two target sequences. In anotherembodiment, the vector comprises three target sequences. In anotherembodiment, the vector comprises four target sequences. In anotherembodiment, the vector comprises five target sequences. In anotherembodiment, the vector comprises six target sequences. In anotherembodiment, the vector comprises seven target sequences. In anotherembodiment, the vector comprises eight target sequences. In otherembodiments, the vector comprises nine, ten, eleven, twelve, thirteen,fourteen, fifteen, or more target sequences.

In one embodiment, the nucleic acid vector is a viral vector. In oneembodiment, the viral vector is an adenoviral vector. In anotherembodiment, the viral vector is a lentiviral vector. In one embodiment,the vector is a retroviral vector. In another embodiment, is anoncoviral vector. Examples of adenoviral vectors include vectors derivedfrom adenoviruses such as, but not limited to, adenovirus type 2 (Ad2),adenovirus type 5 (Ad5), adenovirus type 7 (Ad7) and adenovirus type 12(Ad12). In a further embodiment, the vector is an adeno-associatedvector. Examples of adeno-associated vector includes vectors derivedfrom adeno-associated viruses such as, but not limited to,adeno-associated virus type 1 (AAV1), adeno-associated virus type 2(AAV2), adeno-associated virus type 4 (AAV4), adeno-associated virustype 5 (AAV5), adeno-associated virus type 6 (AAV6), adeno-associatedvirus type 7 (AAV7), and adeno-associated virus type 2 (AAV2). Examplesof lentiviral vectors include vectors derived from lentiviruses such as,but not limited to, HIV-1 and HIV-2. Examples of retroviral vectorsinclude vectors derived from retroviruses such as, but not limited to,Moloney murine leukemia virus (MMLV). Examples of oncoviral vectorsinclude vectors derived from oncoviruses such as, but not limited to,Murine Leukemia Virus (MLV), Spleen Necrosis Virus (SNV), Rous sarcomavirus (RSV) and Avian Leukosis Virus (ALV).

In another embodiment, the vector is delivered to a cell of interest.Delivery can be conducted by any method known to one of skill in theart, including, but not limited to, injection, transfection,lipofection, microinjection, calcium phosphate or calcium chlorideprecipitation, DEAE-dextrin-mediated transfection, or electroporation.Electroporation is carried out at approximate voltage and capacitance toresult in entry of the DNA construct(s) into cells of interest. Othermethods used to transfect cells can also include modified calciumphosphate precipitation, polybrene precipitation, liposome fusion, andreceptor-mediated gene delivery.

In one embodiment, the cell of interest is an endothelial cell. Inanother embodiment, the cell of interest is a vascular smooth musclecell. In further embodiments, the cell may be from the germ line orsomatic, totipotent or pluripotent, dividing or non-dividing, parenchymaor epithelium, immortalized or transformed, or the like. The cell may bea stem cell or a differentiated cell. Cell types that are differentiatedinclude, but are not limited to, adipocytes, fibroblasts, myocytes,cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes,lymphocytes, macrophages, neutrophils, eosinophils, basophils, mastcells, leukocytes, granulocytes, keratinocytes, chondrocytes,osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine andexocrine glands.

In another embodiment, the cell of interest is a cancer cell. The cancercan be, but is not limited to, breast cancer, lung cancer, kidneycancer, brain cancer, liver cancer, colorectal cancers, progressive lungadenocarcinoma, lymphomas, leukemias, adenocarcinomas and sarcomas.

In another aspect, the invention provides an expression systemcomprising a nucleic acid vector, wherein the nucleic acid vectorcomprises a gene of interest and one or more target sequences for amicroRNA within the 3′ UTR region of the gene of interest.

In one embodiment, the gene of interest is a human gene. In anotherembodiment, the gene of interest is a non-human gene. In one embodiment,the gene of interest is a p27 gene. In another embodiment, the gene ofinterest is p53. In other embodiments, the gene of interest is a tumorsuppressor gene. A tumor suppressor gene can include, but is not limitedto, APC, RB1, INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4.

In one embodiment, the microRNA is miR-126. In another embodiment, themicro-RNA is miR-143. In another embodiment, the micro-RNA is miR-145.In further embodiments, the microRNA is Let7-f, miR-27b, miR-130a,miR-221, miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a,miR-378, miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the one or more target sequences comprise SEQ IDNO:2. In another embodiment, the one or more target sequences do notcomprise SEQ ID NO:2. In another embodiment, the one or more targetsequences comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the one or more target sequences do not comprisea nucleic acid sequence complementary to SEQ ID NO:2.

In one embodiment, the vector comprises four target sequences for amicroRNA. In another embodiment, the vector comprises three targetsequences. In another embodiment, the vector comprises four targetsequences. In another embodiment, the vector comprises five targetsequences. In another embodiment, the vector comprises six targetsequences. In another embodiment, the vector comprises seven targetsequences. In another embodiment, the vector comprises eight targetsequences. In other embodiments, the vector comprises nine, ten, eleven,twelve, thirteen, fourteen, fifteen, or more target sequences.

In one embodiment, the target sequences for a microRNA are identical. Inanother embodiment, the target sequences for a microRNA are notidentical. The nucleic acid vectors can comprise different combinationsof target sequences for various microRNAs, including, but not limitedto, miR-126, miR-143, miR-145, Let7-f, miR-27b, miR-130a, miR-221,miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a, miR-378,miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the target sequences for a microRNA comprise SEQ IDNO:2. In another embodiment, the target sequences for a microRNA do notcomprise SEQ ID NO:2. In another embodiment, the target sequences for amicroRNA comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the target sequences for a microRNA do notcomprise a nucleic acid sequence complementary to SEQ ID NO:2.

In another embodiment, the vector comprises a second gene of interest.In one embodiment, the second gene of interest is a human gene. Inanother embodiment, the second gene of interest is a non-human gene. Inone embodiment, the second gene of interest is p53. In otherembodiments, the second gene of interest is a tumor suppressor gene. Atumor suppressor gene can include, but is not limited to, APC, RB1,INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4. In one embodiment, the secondgene of interest is an antithrombotic gene. In another embodiment, thesecond gene of interest is an anti-inflammatory gene. In anotherembodiment, the second gene of interest is ENTPD1, TFPI or PTGIS.

In another embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In one embodiment, the vector comprises one target sequence. In anotherembodiment, the vector comprises two target sequences. In anotherembodiment, the vector comprises three target sequences. In anotherembodiment, the vector comprises four target sequences. In anotherembodiment, the vector comprises five target sequences. In anotherembodiment, the vector comprises six target sequences. In anotherembodiment, the vector comprises seven target sequences. In anotherembodiment, the vector comprises eight target sequences. In otherembodiments, the vector comprises nine, ten, eleven, twelve, thirteen,fourteen, fifteen, or more target sequences.

In one embodiment, the nucleic acid vector is a viral vector. In oneembodiment, the viral vector is an adenoviral vector. In anotherembodiment, the viral vector is a lentiviral vector. In one embodiment,the vector is a retroviral vector. In another embodiment, is anoncoviral vector. Examples of adenoviral vectors include vectors derivedfrom adenoviruses such as, but not limited to, adenovirus type 2 (Ad2),adenovirus type 5 (Ad5), adenovirus type 7 (Ad7) and adenovirus type 12(Ad12). In a further embodiment, the vector is an adeno-associatedvector. Examples of adeno-associated vector includes vectors derivedfrom adeno-associated viruses such as, but not limited to,adeno-associated virus type 1 (AAV1), adeno-associated virus type 2(AAV2), adeno-associated virus type 4 (AAV4), adeno-associated virustype 5 (AAV5), adeno-associated virus type 6 (AAV6), adeno-associatedvirus type 7 (AAV7), and adeno-associated virus type 2 (AAV2). Examplesof lentiviral vectors include vectors derived from lentiviruses such as,but not limited to, HIV-1 and HIV-2. Examples of retroviral vectorsinclude vectors derived from retroviruses such as, but not limited to,Moloney murine leukemia virus (MMLV). Examples of oncoviral vectorsinclude vectors derived from oncoviruses such as, but not limited to,Murine Leukemia Virus (MLV), Spleen Necrosis Virus (SNV), Rous sarcomavirus (RSV) and Avian Leukosis Virus (ALV).

In another aspect, the invention provides a cell comprising a nucleicacid vector, wherein the nucleic acid vector comprises a gene ofinterest and one or more target sequences for a microRNA within the 3′UTR region of the gene of interest.

In one embodiment, the gene of interest is a human gene. In anotherembodiment, the gene of interest is a non-human gene. In one embodiment,the gene of interest is a p27 gene. In another embodiment, the gene ofinterest is p53. In other embodiments, the gene of interest is a tumorsuppressor gene. A tumor suppressor gene can include, but is not limitedto, APC, RB1, INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4.

In one embodiment, the microRNA is miR-126. In another embodiment, themicro-RNA is miR-143. In another embodiment, the micro-RNA is miR-145.In further embodiments, the microRNA is Let7-f, miR-27b, miR-130a,miR-221, miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a,miR-378, miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the one or more target sequences comprise SEQ IDNO:2. In another embodiment, the one or more target sequences do notcomprise SEQ ID NO:2. In another embodiment, the one or more targetsequences comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the one or more target sequences do not comprisea nucleic acid sequence complementary to SEQ ID NO:2.

In one embodiment, the vector comprises four target sequences for amicroRNA. In another embodiment, the vector comprises three targetsequences. In another embodiment, the vector comprises four targetsequences. In another embodiment, the vector comprises five targetsequences. In another embodiment, the vector comprises six targetsequences. In another embodiment, the vector comprises seven targetsequences. In another embodiment, the vector comprises eight targetsequences. In other embodiments, the vector comprises nine, ten, eleven,twelve, thirteen, fourteen, fifteen, or more target sequences.

In one embodiment, the target sequences for a microRNA are identical. Inanother embodiment, the target sequences for a microRNA are notidentical. The nucleic acid vectors can comprise different combinationsof target sequences for various microRNAs, including, but not limitedto, miR-126, miR-143, miR-145, Let7-f, miR-27b, miR-130a, miR-221,miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a, miR-378,miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the target sequences for a microRNA comprise SEQ IDNO:2. In another embodiment, the target sequences for a microRNA do notcomprise SEQ ID NO:2. In another embodiment, the target sequences for amicroRNA comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the target sequences for a microRNA do notcomprise a nucleic acid sequence complementary to SEQ ID NO:2.

In another embodiment, the vector comprises a second gene of interest.In one embodiment, the second gene of interest is a human gene. Inanother embodiment, the second gene of interest is a non-human gene. Inone embodiment, the second gene of interest is p53. In otherembodiments, the second gene of interest is a tumor suppressor gene. Atumor suppressor gene can include, but is not limited to, APC, RB1,INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4. In one embodiment, the secondgene of interest is an antithrombotic gene. In another embodiment, thesecond gene of interest is an anti-inflammatory gene. In anotherembodiment, the second gene of interest is ENTPD1, TFPI or PTGIS.

In another embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In one embodiment, the vector comprises one target sequence. In anotherembodiment, the vector comprises two target sequences. In anotherembodiment, the vector comprises three target sequences. In anotherembodiment, the vector comprises four target sequences. In anotherembodiment, the vector comprises five target sequences. In anotherembodiment, the vector comprises six target sequences. In anotherembodiment, the vector comprises seven target sequences. In anotherembodiment, the vector comprises eight target sequences. In otherembodiments, the vector comprises nine, ten, eleven, twelve, thirteen,fourteen, fifteen, or more target sequences.

In one embodiment, the nucleic acid vector is a viral vector. In oneembodiment, the viral vector is an adenoviral vector. In anotherembodiment, the viral vector is a lentiviral vector. In one embodiment,the vector is a retroviral vector. In another embodiment, is anoncoviral vector. Examples of adenoviral vectors include vectors derivedfrom adenoviruses such as, but not limited to, adenovirus type 2 (Ad2),adenovirus type 5 (Ad5), adenovirus type 7 (Ad7) and adenovirus type 12(Ad12). In a further embodiment, the vector is an adeno-associatedvector. Examples of adeno-associated vector includes vectors derivedfrom adeno-associated viruses such as, but not limited to,adeno-associated virus type 1 (AAV1), adeno-associated virus type 2(AAV2), adeno-associated virus type 4 (AAV4), adeno-associated virustype 5 (AAV5), adeno-associated virus type 6 (AAV6), adeno-associatedvirus type 7 (AAV7), and adeno-associated virus type 2 (AAV2). Examplesof lentiviral vectors include vectors derived from lentiviruses such as,but not limited to, HIV-1 and HIV-2. Examples of retroviral vectorsinclude vectors derived from retroviruses such as, but not limited to,Moloney murine leukemia virus (MMLV). Examples of oncoviral vectorsinclude vectors derived from oncoviruses such as, but not limited to,Murine Leukemia Virus (MLV), Spleen Necrosis Virus (SNV), Rous sarcomavirus (RSV) and Avian Leukosis Virus (ALV).

In another embodiment, the cell expresses the microRNA. In anotherembodiment, the cell does not express the microRNA.

In one embodiment, the gene of interest is not expressed in the cell. Inanother embodiment, the gene of interest is expressed in the cell. Inone embodiment, the second gene of interest is not expressed in thecell. In another embodiment, the second gene of interest is expressed inthe cell.

In one embodiment, the cell is an endothelial cell. In anotherembodiment, the cell is a vascular smooth muscle cell. In furtherembodiments, the cell may be from the germ line or somatic, totipotentor pluripotent, dividing or non-dividing, parenchyma or epithelium,immortalized or transformed, or the like. The cell may be a stem cell ora differentiated cell. Cell types that are differentiated include, butare not limited to, adipocytes, fibroblasts, myocytes, cardiomyocytes,endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes,macrophages, neutrophils, eosinophils, basophils, mast cells,leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts,osteoclasts, hepatocytes, and cells of the endocrine and exocrineglands.

In another embodiment, the cell is a cancer cell. In one embodiment, thecancer is, but is not limited to, breast cancer, lung cancer, kidneycancer, brain cancer, liver cancer, colorectal cancers, progressive lungadenocarcinoma, lymphomas, leukemias, adenocarcinomas and sarcomas.

In other embodiments, the nucleic acid vector is delivered into the cellby any method known to one of skill in the art, such as, but not limitedto, injection, transfection, lipofection, microinjection, calciumphosphate or calcium chloride precipitation, DEAE-dextrin-mediatedtransfection, or electroporation. Electroporation is carried out atapproximate voltage and capacitance to result in entry of the DNAconstruct(s) into cells of interest. Other methods used to transfectcells can also include modified calcium phosphate precipitation,polybrene precipitation, liposome fusion, and receptor-mediated genedelivery.

DNA and Amino Acid Manipulation Methods and Purification Thereof

The present invention utilizes conventional molecular biology,microbiology, and recombinant DNA techniques available to one ofordinary skill in the art. Such techniques are well known to the skilledworker and are explained fully in the literature. See, e.g., Maniatis,Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual” (1982):“DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover,ed., 1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984);“Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985);“Transcription and Translation” (B. D. Hames & S. J. Higgins, eds.,1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “ImmobilizedCells and Enzymes” (IRL Press, 1986): B. Perbal, “A Practical Guide toMolecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: aLaboratory Manual” (1989).

MicroRNAs

Nucleic acid sequences of various microRNAs, and nucleic acid sequencesof various target sequences for microRNAs, are available in the art.

The nucleic acid sequence of miR-126 is depicted in SEQ ID NO: 1.

SEQ ID NO: 1 is a sequence of human miR-126 (residues 1-22)

UCGUACCGUGAGUAAUAAUGCG

The nucleic acid sequence of a target sequence of miR-126 is depicted inSEQ ID NO: 2.

SEQ ID NO: 2 is a nucleic acid target sequence of human miR-126(residues 1-22)

CGCATTATTACTCACGGTACGAOne of skill in the art can determine the complementary sequence of anucleic acid target sequence of a microRNA, such as, but not limited to,SEQ ID NO. 2.

Proteins

One skilled in the art can obtain a protein in several ways, whichinclude, but are not limited to, isolating the protein via biochemicalmeans or expressing a nucleotide sequence encoding the protein ofinterest by genetic engineering methods.

A protein is encoded by a nucleic acid (including, for example, genomicDNA, complementary DNA (cDNA), synthetic DNA, as well as any form ofcorresponding RNA). For example, it can be encoded by a recombinantnucleic acid of a gene. The proteins of the invention can be obtainedfrom various sources and can be produced according to various techniquesknown in the art. For example, a nucleic acid that encodes a protein canbe obtained by screening DNA libraries, or by amplification from anatural source. A protein can be a fragment or portion thereof. Thenucleic acids encoding a protein can be produced via recombinant DNAtechnology and such recombinant nucleic acids can be prepared byconventional techniques, including chemical synthesis, geneticengineering, enzymatic techniques, or a combination thereof. Forexample, a p27 protein is the polypeptide encoded by the nucleic acidhaving the nucleotide sequence shown in SEQ ID NO: 4. An example of anp27 polypeptide has the amino acid sequence shown in SEQ ID NO: 3.

The polypeptide sequence of human p27 is depicted in SEQ ID NO: 3. Thenucleotide sequence of human p27 is shown in SEQ ID NO: 4. Sequenceinformation related to p27 is accessible in public databases by GenBankAccession numbers NM_(—)004064 (for nucleic acid) and NP_(—)004055 (forprotein).

SEQ ID NO: 3 is the human wild type amino acid sequence corresponding top27 (residues 1-198)

msnvrvsngs pslermdarq aehpkpsacr nlfgpvdheeltrdlekhcr dmeeasqrkw nfdfqnhkpl egkyewqevekgslpefyyr pprppkgack vpaqesqdvs gsrpaapligapansedthl vdpktdpsds qtglaeqcag irkrpatddsstqnkranrt eenvsdgspn agsveqtpkk pglrrrqt

SEQ ID NO: 4 is the human wild type nucleic acid sequence correspondingto p27 (residues 1-2413)

1 cttcttcgtc agcctccctt ccaccgccat attgggccac taaaaaaagg gggctcgtct 61tttcggggtg tttttctccc cctcccctgt ccccgcttgc tcacggctct gcgactccga 121cgccggcaag gtttggagag cggctgggtt cgcgggaccc gcgggcttgc acccgcccag 181actcggacgg gctttgccac cctctccgct tgcctggtcc cctctcctct ccgccctccc 241gctcgccagt ccatttgatc agcggagact cggcggccgg gccggggctt ccccgcagcc 301cctgcgcgct cctagagctc gggccgtggc tcgtcggggt ctgtgtcttt tggctccgag 361ggcagtcgct gggcttccga gaggggttcg ggctgcgtag gggcgctttg ttttgttcgg 421ttttgttttt ttgagagtgc gagagaggcg gtcgtgcaga cccgggagaa agatgtcaaa 481cgtgcgagtg tctaacggga gccctagcct ggagcggatg gacgccaggc aggcggagca 541ccccaagccc tcggcctgca ggaacctctt cggcccggtg gaccacgaag agttaacccg 601ggacttggag aagcactgca gagacatgga agaggcgagc cagcgcaagt ggaatttcga 661ttttcagaat cacaaacccc tagagggcaa gtacgagtgg caagaggtgg agaagggcag 721cttgcccgag ttctactaca gacccccgcg gccccccaaa ggtgcctgca aggtgccggc 781gcaggagagc caggatgtca gcgggagccg cccggcggcg cctttaattg gggctccggc 841taactctgag gacacgcatt tggtggaccc aaagactgat ccgtcggaca gccagacggg 901gttagcggag caatgcgcag gaataaggaa gcgacctgca accgacgatt cttctactca 961aaacaaaaga gccaacagaa cagaagaaaa tgtttcagac ggttccccaa atgccggttc 1021tgtggagcag acgcccaaga agcctggcct cagaagacgt caaacgtaaa cagctcgaat 1081taagaatatg tttccttgtt tatcagatac atcactgctt gatgaagcaa ggaagatata 1141catgaaaatt ttaaaaatac atatcgctga cttcatggaa tggacatcct gtataagcac 1201tgaaaaacaa caacacaata acactaaaat tttaggcact cttaaatgat ctgcctctaa 1261aagcgttgga tgtagcatta tgcaattagg tttttcctta tttgcttcat tgtactacct 1321gtgtatatag tttttacctt ttatgtagca cataaacttt ggggaaggga gggcagggtg 1381gggctgagga actgacgtgg agcggggtat gaagagcttg ctttgattta cagcaagtag 1441ataaatattt gacttgcatg aagagaagca attttgggga agggtttgaa ttgttttctt 1501taaagatgta atgtcccttt cagagacagc tgatacttca tttaaaaaaa tcacaaaaat 1561ttgaacactg gctaaagata attgctattt atttttacaa gaagtttatt ctcatttggg 1621agatctggtg atctcccaagactatctaaag tttgttagat agctgcatgt ggctttttta 1681aaaaagcaac agaaacctat cctcactgcc ctccccagtc tctcttaaag ttggaattta 1741ccagttaatt actcagcaga atggtgatca ctccaggtag tttggggcaa aaatccgagg 1801tgcttgggag ttttgaatgt taagaattga ccatctgctt ttattaaatt tgttgacaaa 1861attttctcat tttcttttca cttcgggctg tgtaaacaca gtcaaaataa ttctaaatcc 1921ctcgatattt ttaaagatct gtaagtaact tcacattaaa aaatgaaata ttttttaatt 1981taaagcttac tctgtccatt tatccacagg aaagtgttat ttttcaagga aggttcagtg 2041agagaaaagc acacttgtag gataagtgaa atggatacta catctttaaa cagtatttca 2101ttgcctgtgt atggaaaaac catttgaagt gtacctgtgt acataactct gtaaaaacac 2161tgaaaaatta tactaactta tttatgttaa aagatttttt ttaatctaga caatatacaa 2221gccaaagtgg catgttttgt gcatttgtaa atgctgtgtt gggtagaata ggttttcccc 2281tcttttgtta aataatatgg ctatgcttaa aaggttgcat actgagccaa gtataatttt 2341ttgtaatgtg tgaaaaagat gccaattatt gttacacatt aagtaatcaa taaagaaaac 2401ttccatagct att

p27 is a protein encoded by the CDKN1B gene. p27 is a cyclin-dependentkinase inhibitor, and is also known as p27^(Kip1). It is a member of thekinase inhibitor protein (KIP) family. For additional information onp27, see e.g., Nisar P. Malek, Holly Sundberg, Seth McGrew, KeikoNakayama. Themis R. Kyriakidis & James M. Roberts, 2001, A mouseknock-in model exposes sequential proteolytic pathways that regulatep27Kip1 in G1 and S phase, Nature, 413, 323-327.

Protein Variants

Protein variants can include amino acid sequence modifications. Forexample, amino acid sequence modifications fall into one or more ofthree classes: substitutional, insertional or deletional variants.Insertions can include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. These variants ordinarilyare prepared by site-specific mutagenesis of nucleotides in the DNAencoding the protein, thereby producing DNA encoding the variant, andthereafter expressing the DNA in recombinant cell culture.

Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example M13 primermutagenesis and PCR mutagenesis. Amino acid substitutions can be singleresidues, but can occur at a number of different locations at once. Inone non-limiting embodiment, insertions can be on the order of aboutfrom 1 to about 10 amino acid residues, while deletions can range fromabout 1 to about 30 residues. Deletions or insertions can be made inadjacent pairs (for example, a deletion of about 2 residues or insertionof about 2 residues). Substitutions, deletions, insertions, or anycombination thereof can be combined to arrive at a final construct. Themutations cannot place the sequence out of reading frame and should notcreate complementary regions that can produce secondary mRNA structure.Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place.

Substantial changes in function or immunological identity are made byselecting residues that differ more significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site or (c)the bulk of the side chain. The substitutions that can produce thegreatest changes in the protein properties will be those in which (a) ahydrophilic residue, e.g. seryl or threonyl, is substituted for (or by)a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl oralanyl; (b) a cysteine or proline is substituted for (or by) any otherresidue: (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine, in this case, (e) byincreasing the number of sites for sulfation and/or glycosylation.

Minor variations in the amino acid sequences of proteins are provided bythe present invention. The variations in the amino acid sequence can bewhen the sequence maintains at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about75%, at least about 80%, at least about 90%, at least about 95%, or atleast about 99% identity to SEQ ID NO: 3, or any other amino acidsequence of interest. For example, conservative amino acid replacementscan be utilized. Conservative replacements are those that take placewithin a family of amino acids that are related in their side chains,wherein the interchangeability of residues have similar side chains.

Genetically encoded amino acids are generally divided into families: (1)acidic amino acids are aspartate, glutamate: (2) basic amino acids arelysine, arginine, histidine; (3) non-polar amino acids are alanine,valine, leucine, isoleucine, proline, phenylalanine, methionine,tryptophan, and (4) uncharged polar amino acids are glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic aminoacids include arginine, asparagine, aspartate, glutamine, glutamate,histidine, lysine, serine, and threonine. The hydrophobic amino acidsinclude alanine, cysteine, isoleucine, leucine, methionine,phenylalanine, proline, tryptophan, tyrosine and valine. Other familiesof amino acids include (i) a group of amino acids havingaliphatic-hydroxyl side chains, such as serine and threonine; (ii) agroup of amino acids having amide-containing side chains, such asasparagine and glutamine; (iii) a group of amino acids having aliphaticside chains such as glycine, alanine, valine, leucine, and isoleucine;(iv) a group of amino acids having aromatic side chains, such asphenylalanine, tyrosine, and tryptophan; and (v) a group of amino acidshaving sulfur-containing side chains, such as cysteine and methionine.Useful conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine valine, glutamic-aspartic, and asparagine-glutamine.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gin; Ser,Thr; Lys, Arg; and Phe, Tyr. Substitutional or deletional mutagenesiscan be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) orO-glycosylation (Ser or Thr). Deletions of cysteine or other labileresidues also can be desirable. Deletions or substitutions of potentialproteolysis sites, e.g. Arg, is accomplished for example by deleting oneof the basic residues or substituting one by glutaminyl or histidylresidues.

Bacterial and Yeast Expression Systems

In bacterial systems, a number of expression vectors can be selected.For example, when a large quantity of a protein encoded by a gene isneeded for the induction of antibodies, vectors which direct high levelexpression of proteins that are readily purified can be used.Non-limiting examples of such vectors include multifunctional E. colicloning and expression vectors such as BLUESCRIPT (Stratagene). pINvectors or pGEX vectors (Promega, Madison, Wis.) also can be used toexpress foreign polypeptide molecules as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toglutathione-agarose beads followed by elution in the presence of freeglutathione. Proteins made in such systems can be designed to includeheparin, thrombin, or factor Xa protease cleavage sites so that thecloned polypeptide of interest can be released from the GST moiety atwill.

Plant and Insect Expression Systems

If plant expression vectors are used, the expression of sequencesencoding a protein can be driven by any of a number of promoters. Forexample, viral promoters such as the 35S and 19S promoters of CaMV canbe used alone or in combination with the omega leader sequence from TMV.Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters, can be used. These constructs can be introducedinto plant cells by direct DNA transformation or by pathogen-mediatedtransfection.

An insect system also can be used to express proteins. For example, inone such system Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. Sequences encoding apolypeptide can be cloned into a non-essential region of the virus, suchas the polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of nucleic acid sequences, such as asequence corresponding to a gene of interest, will render the polyhedringene inactive and produce recombinant virus lacking coat protein. Therecombinant viruses can then be used to infect S. frugiperda cells orTrichoplusia larvae in which the protein or a variant thereof can beexpressed.

Mammalian Expression Systems

An expression vector can include a nucleotide sequence that encodes apolypeptide linked to at least one regulatory sequence in a mannerallowing expression of the nucleotide sequence in a host cell. A numberof viral-based expression systems can be used to express a protein or avariant thereof in mammalian host cells. For example, if an adenovirusis used as an expression vector, sequences encoding a protein can beligated into an adenovirus transcription/translation complex comprisingthe late promoter and tripartite leader sequence. Insertion into anon-essential E1 or E3 region of the viral genome can be used to obtaina viable virus which expresses a protein in infected host cells.Transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,can also be used to increase expression in mammalian host cells.

Regulatory sequences are well known in the art, and can be selected todirect the expression of a protein or polypeptide of interest in anappropriate host cell as described in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Non-limiting examples of regulatory sequences include:polyadenylation signals, promoters (such as CMV, ASV, SV40, or otherviral promoters such as those derived from bovine papilloma, polyoma,and Adenovirus 2 viruses (Fiers, et al., 1973, Nature 273:113; Hager GL, et al., Curr Opin Genet Dev, 2002, 12(2):137-41) enhancers, and otherexpression control elements.

Enhancer regions, which are those sequences found upstream or downstreamof the promoter region in non-coding DNA regions, are also known in theart to be important in optimizing expression. If needed, origins ofreplication from viral sources can be employed, such as if a prokaryotichost is utilized for introduction of plasmid DNA. However, in eukaryoticorganisms, chromosome integration is a common mechanism for DNAreplication.

Suitable promoters for expressing RNA from a plasmid include, e.g., theU6 or H1 RNA pol III promoter sequences, or the cytomegaloviruspromoters. Selection of other suitable promoters is within the skill inthe art. Recombinant plasmids can comprise inducible or regulatablepromoters for expression of the RNA in cells (such as, but not limitedto, vascular smooth muscle cells or endothelial cells). For example, anucleic acid encoding a gene, such as, but not limited to, a p27 gene,and one or more target sequences for a microRNA, such as, but notlimited to, miR-126 (comprising one or more sequences of SEQ ID NO: 2)can be placed under the control of the CMV intermediate-early promoter,whereby the nucleic acid sequences encoding the p27 gene are located 3′of the promoter, so that the promoter can initiate transcription of themiRNA gene product coding sequences.

A gene that encodes a selectable marker (for example, resistance toantibiotics or drugs, such as ampicillin, neomycin, G418, andhygromycin) can be introduced into host cells along with the gene ofinterest in order to identify and select clones that stably express agene encoding a protein of interest. The gene encoding a selectablemarker can be introduced into a host cell on the same plasmid as thegene of interest or can be introduced on a separate plasmid. Cellscontaining the gene of interest can be identified by drug selectionwherein cells that have incorporated the selectable marker gene willsurvive in the presence of the drug. Cells that have not incorporatedthe gene for the selectable marker die. Surviving cells can then bescreened for the production of the desired protein molecule.

For stable transfection of mammalian cells, a small fraction of cellscan integrate introduced DNA into their genomes. The expression vectorand transfection method utilized can be factors that contribute to asuccessful integration event. For stable amplification and expression ofa desired protein, a vector containing DNA encoding a protein ofinterest is stably integrated into the genome of eukaryotic cells (forexample mammalian cells, such as, but not limited to, vascular smoothmuscle cells or endothelial cells), resulting in the stable expressionof transfected genes. An exogenous nucleic acid sequence can beintroduced into a cell (such as a mammalian cell, either a primary orsecondary cell) by homologous recombination as disclosed in U.S. Pat.No. 5,641,670, the contents of which are herein incorporated byreference.

Vectors

Nucleic acid vectors, such as plasmids, suitable for expressing genes ofinterest and target sequences for microRNAs, methods for insertingnucleic acid sequences into the plasmid to express the gene of interest,and methods of delivering the recombinant vectors to cells of interestare well-established and practiced in the art. See, for example, Zeng etal. (2002), Molecular Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol,20:446-448; Brummelkamp et al. (2002). Science 296:550-553; Miyagishi etal. (2002), Nat. Biotechnol. 20:497-500; Paddison et al. (2002), GenesDev. 16:948-958; Lee et al. (2002), Nat. Biotechnol. 20:500-505; andPaul et al. (2002), Nat. Biotechnol. 20:505-508, the entire disclosuresof which are herein incorporated by reference.

Genes of interest and target sequences for microRNAs can also beexpressed from recombinant viral vectors. The RNA expressed from therecombinant viral vectors can either be isolated from cultured cellexpression systems by standard techniques, or can be expressed directlyin mammalian cells (for example, vascular smooth muscle cells orendothelial cells). For example, the recombinant viral vectors cancomprise sequences that encode the gene of interest and any suitablepromoter for expressing the RNA sequences. Vectors can also compriseinducible or regulatable promoters for expression of the gene ofinterest in cells, such as mammalian cells. As discussed previously,non-limiting examples of suitable promoters include the U6 or H1 RNA polIII promoter sequences, or the cytomegalovirus promoters. Selection ofother suitable promoters is practiced by those of ordinary skill in theart.

Any viral vector that can harbor the nucleotide sequences for the geneof interest and the one or more target sequences for a microRNA of theinvention can be used. Non-limiting examples of such vectors include:vectors derived from adenovirus (AV); adeno-associated virus (AAV);retroviruses (e.g., lentiviruses (LV), Rhabdoviruses, murine leukemiavirus); herpes virus, and the like. The tropism of the viral vectors canbe modified by pseudotyping the vectors with envelope proteins or othersurface antigens from other viruses, or by substituting different viralcapsid proteins, as appropriate. For example, lentiviral vectors can bepseudotyped with surface proteins from vesicular stomatitis virus (VSV),rabies, Ebola, Mokola, and the like. For example, AAV vectors can bemade to target different cells by engineering the vectors to expressdifferent capsid protein serotypes. An AAV vector expressing a serotype2 capsid on a serotype 2 genome is called AAV 2/2. This serotype 2capsid gene in the AAV 2/2 vector can be replaced by a serotype 5 capsidgene to produce an AAV 2/5 vector. Techniques for constructing AAVvectors which express different capsid protein serotypes are within theskill in the art; see, e.g., Rabinowitz J. E. et al. (2002), J Virol76:791-801, the entire disclosure of which is herein incorporated byreference.

Recombinant viral vectors suitable for expressing the gene of interestand the one or more target sequences for a microRNA of the invention,methods for inserting nucleic acid sequences for expressing genes ofinterest in the vector, methods of delivering the viral vector to cellsof interest, and recovery of the expressed nucleic acid molecules andproteins are within the skill in the art. See, for example, Dornburg(1995), Gene Therap. 2:301-310; Eglitis (1988), Biotechniques 6:608-614;Miller (1990), Hum. Gene Therap. 1:5-14; and Anderson (1998), Nature392:25-30, the entire disclosures of which are herein incorporated byreference. Useful viral vectors can be those derived from AV and AAV. Asuitable AV vector for expressing a nucleic acid molecule of theinvention, a method for constructing the recombinant AV vector, and amethod for delivering the vector into target cells, are described in Xiaet al. (2002), Nat. Biotech. 20:1006-1010, the entire disclosure ofwhich is herein incorporated by reference. Methods for constructing therecombinant AAV vector, and methods for delivering the vectors intotarget cells are described in Samulski et al. (1987), J. Virol.61:3096-3101; Fisher et al. (1996). J. Virol. 70:520-532; Samulski etal. (1989), J. Virol. 63:3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat.No. 5,139,941; International Patent Application No. WO 94/13788: andInternational Patent Application No. WO 93/24641, the entire disclosuresof which are herein incorporated by reference.

Cell Delivery

A eukaryotic expression vector can be used to transfect cells in orderto produce proteins encoded by nucleotide sequences of the vector.Mammalian cells (such as, but not limited to, vascular smooth musclecells or endothelial cells) can contain an expression vector (forexample, one that contains a gene encoding a p27 protein or polypeptide)via introducing the expression vector into an appropriate host cell viamethods known in the art.

A host cell strain can be chosen for its ability to modulate theexpression of the inserted sequences or to process the expressedpolypeptide encoded by a gene, such as a p27 gene, in the desiredfashion. Such modifications of the polypeptide include, but are notlimited to, acetylation, carboxylation, glycosylation, phosphorylation,lipidation, and acylation. Post-translational processing which cleaves a“prepro” form of the polypeptide also can be used to facilitate correctinsertion, folding and/or function. Different host cells which havespecific cellular machinery and characteristic mechanisms forpost-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38),are available from the American Type Culture Collection (ATCC: 10801University Boulevard, Manassas, Va. 20110-2209) and can be chosen toensure the correct modification and processing of the foreign protein.

An exogenous nucleic acid can be introduced into a cell via a variety oftechniques known in the art, such as lipofection, microinjection,calcium phosphate or calcium chloride precipitation.DEAE-dextran-mediated transfection, or electroporation. Electroporationis carried out at approximate voltage and capacitance to result in entryof the DNA construct(s) into cells of interest (such as cells of the endbulb of a hair follicle, for example dermal papilla cells or dermalsheath cells). Other transfection methods also include modified calciumphosphate precipitation, polybrene precipitation, liposome fusion, andreceptor-mediated gene delivery.

Cells that will be genetically engineered can be primary and secondarycells obtained from various tissues, and include cell types which can bemaintained and propagated in culture. Vertebrate tissue can be obtainedby methods known to one skilled in the art, such a punch biopsy or othersurgical methods of obtaining a tissue source of the primary cell typeof interest. A mixture of primary cells can be obtained from the tissue,using methods readily practiced in the art, such as explanting orenzymatic digestion (for examples using enzymes such as pronase,trypsin, collagenase, elastase dispase, and chymotrypsin). Biopsymethods have also been described in United States Patent ApplicationPublication 2004/0057937 and PCT application publication WO 2001/32840,and are hereby incorporated by reference.

Primary cells can be acquired from the individual to whom thegenetically engineered primary or secondary cells are administered.However, primary cells can also be obtained from a donor, other than therecipient, of the same species. The cells can also be obtained fromanother species (for example, rabbit, cat, mouse, rat, sheep, goat, dog,horse, cow, bird, or pig). Primary cells can also include cells from anisolated vertebrate tissue source grown attached to a tissue culturesubstrate (for example, flask or dish) or grown in a suspension; cellspresent in an explant derived from tissue; both of the aforementionedcell types plated for the first time; and cell culture suspensionsderived from these plated cells. Secondary cells can be plated primarycells that are removed from the culture substrate and replated, orpassaged, in addition to cells from the subsequent passages. Secondarycells can be passaged one or more times. These primary or secondarycells can contain expression vectors having a gene that encodes aprotein of interest (for example, a p27 protein or polypeptide).

Delivery of nucleic acids into viable cells can be effected ex vivo, insitu, or in vivo by use of vectors, and more particularly viral vectors(e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus),or ex vivo by use of physical DNA transfer methods (e.g., liposomes orchemical treatments). Non-limiting techniques suitable for the transferof nucleic acid into mammalian cells in vitro include the use ofliposomes, electroporation, microinjection, cell fusion, DEAE-dextran,and the calcium phosphate precipitation method (see, for example,Anderson, Nature, supplement to vol. 392, no. 6679, pp. 25-20 (1998)).Introduction of a nucleic acid or a gene encoding a polypeptide of theinvention can also be accomplished with extrachromosomal substrates(transient expression) or artificial chromosomes (stable expression).Cells may also be cultured ex vivo in the presence of therapeuticcompositions of the present invention in order to proliferate or toproduce a desired effect on or activity in such cells. Treated cells canthen be introduced in vivo for therapeutic purposes.

Nucleic acids can be inserted into vectors and used as gene therapyvectors. A number of viruses have been used as gene transfer vectors,including papovaviruses, e.g., SV40 (Madzak et al., (1992) J Gen Virol.73(Pt 6):1533-6), adenovirus (Berkner (1992) Curr Top Microbiol Immunol.158:39-66; Berkner (1988) Biotechniques, 6(7):616-29; Gorziglia andKapikian (1992) J Virol. 66(7):4407-12; Quantin et al., (1992) Proc NatlAcad Sci USA. 89(7):2581-4; Rosenfeld et al., (1992) Cell. 68(1):143-55;Wilkinson et al., (1992) Nucleic Acids Res. 20(9):2233-9;Stratford-Perricaudet et al., (1990) Hum Gene Ther. 1(3):241-56),vaccinia virus (Moss (1992) Curr Opin Biotechnol. 3(5):518-22),adeno-associated virus (Muzyczka, (1992) Curr Top Microbiol Immunol.158:97-129; Ohi et al., (1990) Gene. 89(2):279-82), herpesvirusesincluding HSV and EBV (Margolskee (1992) Curr Top Microbhiol Immunol.158:67-95; Johnson et al., (1992) Brain Res Mol Brain Res.12(1-3):95-102: Fink et al., (1992) Hum Gene Ther. 3(1):11-9:Breakefield and Geller (1987) Mol Neurobiol. 1(4):339-71; Freese et al.,(1990) Biochem Pharmacol. 40(10):2189-99), and retroviruses of avian(Bandyopadhyay and Temin (1984) Mol Cell Biol. 4(4):749-54; Petropouloset al., (1992) J Virol. 66(6):3391-7), murine (Miller et al. (1992) MolCell Biol. 12(7):3262-72; Miller et al., (1985) J Virol. 55(3):521-6;Sorge et al., (1984) Mol Cell Biol. 4(9):1730-7; Mann and Baltimore(1985) J Virol. 54(2):401-7: Miller et al., (1988) J Virol.62(11):4337-45), and human origin (Shimada et al., (1991) J Clin Invest.88(3):1043-7; Helseth et al., (1990) J Virol. 64(12):6314-8; Page etal., (1990) J Virol. 64(11):5270-6; Buchschacher and Panganiban (1992) JVirol. 66(5):2731-9).

Non-limiting examples of in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors (see U.S. Pat.No. 5,252,479, which is incorporated by reference in its entirety) andviral coat protein-liposome mediated transfection (Dzau et al., Trendsin Biotechnology 11:205-210 (1993), incorporated entirely by reference).For example, naked DNA vaccines are generally known in the art; seeBrower, Nature Biotechnology, 16:1304-1305 (1998), which is incorporatedby reference in its entirety. Gene therapy vectors can be delivered to asubject by, for example, intravenous injection, local administration(see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see,e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91: 3054-3057). Thepharmaceutical preparation of the gene therapy vector can include thegene therapy vector in an acceptable diluent, or can comprise a slowrelease matrix in which the gene delivery vehicle is imbedded.Alternatively, where the complete gene delivery vector can be producedintact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

For reviews of gene therapy protocols and methods see Anderson et al.,Science 256:808-813 (1992); U.S. Pat. Nos. 5,252,479, 5,747,469,6,017,524, 6,143,290, 6,410,010 6,511,847; 8,398,968; and 8,404,653which are all hereby incorporated by reference in their entireties. Foran example of gene therapy treatment in humans see Porter et al., NEJM2011 365:725-733 and Kalos et al. Sci. Transl. Med. 2011. 201 3(95):95.For additional reviews of gene therapy technology, see Friedmann.Science, 244:1275-1281 (1989); Verma, Scientific American: 68-84 (1990);Miller, Nature, 357: 455-460 (1992); Kikuchi et al., J Dermatol Sci.2008 May; 50(2):87-98; Isaka et al., Expert Opin Drug Deliv. 2007September; 4(5):561-71; Jager et al., Curr Gene Ther. 2007 August;7(4):272-83; Waehler et al., Nat Rev Genet. 2007 August; 8(8):573-87;Jensen et al., Ann Med. 2007:39(2):108-15; Herweijer et al., Gene Ther.2007 January; 14(2):99-107; Eliyahu et al., Molecules, 2005 Jan. 31;10(1):34-64; and Altaras et al., Adv Biochem Eng Biotechnol. 2005;99:193-260, all of which are hereby incorporated by reference in theirentireties.

Cell Culturing

Various culturing parameters can be used with respect to the host cellbeing cultured. Appropriate culture conditions for mammalian cells arewell known in the art (Cleveland W L, et al., J Immunol Methods, 1983,56(2): 221-234) or can be determined by the skilled artisan (see, forexample, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D.and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cellculturing conditions can vary according to the type of host cellselected. Commercially available medium can be utilized. Non-limitingexamples of medium include, for example, Minimal Essential Medium (MEM,Sigma, St. Louis, Mo.): Dulbecco's Modified Eagles Medium (DMEM, Sigma);Ham's F10 Medium (Sigma); HyClone cell culture medium (HyClone, Logan,Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media,which are formulated for various cell types, e.g., CD-CHO Medium(Invitrogen, Carlsbad, Calif.).

The cell culture media can be supplemented as necessary withsupplementary components or ingredients, including optional components,in appropriate concentrations or amounts, as necessary or desired. Cellculture medium solutions provide at least one component from one or moreof the following categories: (1) an energy source, usually in the formof a carbohydrate such as glucose; (2) all essential amino acids, andusually the basic set of twenty amino acids plus cysteine; (3) vitaminsand/or other organic compounds required at low concentrations; (4) freefatty acids or lipids, for example linoleic acid; and (5) traceelements, where trace elements are defined as inorganic compounds ornaturally occurring elements that can be required at very lowconcentrations, usually in the micromolar range.

The medium also can be supplemented electively with one or morecomponents from any of the following categories: (1) salts, for example,magnesium, calcium, and phosphate: (2) hormones and other growth factorssuch as, serum, insulin, transferrin, and epidermal growth factor: (3)protein and tissue hydrolysates, for example peptone or peptone mixtureswhich can be obtained from purified gelatin, plant material, or animalbyproducts; (4) nucleosides and bases such as, adenosine, thymidine, andhypoxanthine; (5) buffers, such as HEPES: (6) antibiotics, such asgentamycin or ampicillin; (7) cell protective agents, for examplepluronic polyol; and (8) galactose. In one embodiment, soluble factorscan be added to the culturing medium.

The mammalian cell culture that can be used with the present inventionis prepared in a medium suitable for the type of cell being cultured. Inone embodiment, the cell culture medium can be any one of thosepreviously discussed (for example, MEM) that is supplemented with serumfrom a mammalian source (for example, fetal bovine serum (FBS)). Inanother embodiment, the medium can be a conditioned medium to sustainthe growth of mammalian cells. In other embodiments of the invention,cells are grown in a suspension culture (for example, athree-dimensional culture such as a hanging drop culture) in thepresence of an effective amount of enzyme, wherein the enzyme substrateis an extracellular matrix molecule in the suspension culture. Forexample, the enzyme can be a hyaluronidase.

A suspension culture is a type of culture wherein cells, or aggregatesof cells multiply while suspended in liquid medium. A suspension culturecomprising mammalian cells can be used for the maintenance of cell typesthat do not adhere or to enable cells to manifest specific cellularcharacteristics that are not seen in the adherent form. Some types ofsuspension cultures can include three-dimensional cultures or a hangingdrop culture. A hanging-drop culture is a culture in which the materialto be cultivated is inoculated into a drop of fluid attached to a flatsurface (such as a coverglass, glass slide, Petri dish, flask, and thelike), and can be inverted over a hollow surface. Cells in a hangingdrop can aggregate toward the hanging center of a drop as a result ofgravity. However, according to the methods of the invention, cellscultured in the presence of a protein that degrades the extracellularmatrix (such as collagenase, chondroitinase, hyaluronidase, and thelike) will become more compact and aggregated within the hanging dropculture, for degradation of the ECM will allow cells to become closer inproximity to one another since less of the ECM will be present. See alsoInternational PCT Publication No. WO2007/100870, which is incorporatedby reference.

Three-dimensional cultures can be formed from agar (such as Gey's Agar),hydrogels (such as matrigel, agarose, and the like; Lee et al., (2004)Biomaterials 25: 2461-2466) or polymers that are cross-linked. Thesepolymers can comprise natural polymers and their derivatives, syntheticpolymers and their derivatives, or a combination thereof. Naturalpolymers can be anionic polymers, cationic polymers, amphipathicpolymers, or neutral polymers. Non-limiting examples of anionic polymerscan include hyaluronic acid, alginic acid (alginate), carageenan,chondroitin sulfate, dextran sulfate, and pectin. Some examples ofcationic polymers, include but are not limited to, chitosan orpolylysine. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A.S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NYAcad Sci 944: 62-73). Examples of amphipathic polymers can include, butare not limited to collagen, gelatin, fibrin, and carboxymethyl chitin.Non-limiting examples of neutral polymers can include dextran, agarose,or pullulan. (Peppas et al., (2006) Adv Mater. 18: 1345-60; Hoffman, A.S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NYAcad Sci 944: 62-73).

Cells suitable for culturing according to methods of the invention canharbor introduced expression vectors, such as plasmids. The expressionvector constructs can be introduced via transformation, microinjection,transfection, lipofection, electroporation, or infection. The expressionvectors can contain coding sequences, or portions thereof, encoding theproteins for expression and production. Expression vectors containingsequences encoding the produced proteins and polypeptides, as well asthe appropriate transcriptional and translational control elements, canbe generated using methods well known to and practiced by those skilledin the art. These methods include synthetic techniques, in vitrorecombinant DNA techniques, and in vivo genetic recombination which aredescribed in J. Sambrook et al., 2001, Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubelet al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

Obtaining and Purifying Polypeptides

A polypeptide molecule encoded by a gene, such as a p27 gene, or avariant thereof, can be obtained by purification from human cellsexpressing a protein or polypeptide encoded by a p27 gene via in vitroor in vivo expression of a nucleic acid sequence encoding a p27 proteinor polypeptide: or by direct chemical synthesis.

Detecting Polypeptide Expression

Host cells which contain a nucleic acid encoding a p27 protein orpolypeptide, and which subsequently express a protein encoded by a p27gene, can be identified by various procedures known to those of skill inthe art. These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations and protein bioassay or immunoassay techniqueswhich include membrane, solution, or chip-based technologies for thedetection and/or quantification of nucleic acid or protein. For example,the presence of a nucleic acid encoding a p27 protein or polypeptide canbe detected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or fragments of nucleic acids encoding a p27 protein orpolypeptide. In one embodiment, a fragment of a nucleic acid of a p27gene can encompass any portion of at least about 8 consecutivenucleotides of SEQ ID NO: 4. In another embodiment, the fragment cancomprise at least about 10 consecutive nucleotides, at least about 15consecutive nucleotides, at least about 20 consecutive nucleotides, orat least about 30 consecutive nucleotides of SEQ ID NO: 4. Fragments caninclude all possible nucleotide lengths between about 8 and about 100nucleotides, for example, lengths between about 15 and about 100nucleotides, or between about 20 and about 100 nucleotides. Nucleic acidamplification-based assays involve the use of oligonucleotides selectedfrom sequences encoding a polypeptide encoded by a p27 gene to detecttransformants which contain a nucleic acid encoding a p27 protein orpolypeptide.

Protocols for detecting and measuring the expression of a polypeptideencoded by a gene, such as a p27 gene, using either polyclonal ormonoclonal antibodies specific for the polypeptide are well established.Non-limiting examples include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS).A two-site, monoclonal-based immunoassay using monoclonal antibodiesreactive to two non-interfering epitopes on a polypeptide encoded by agene, such as a p27 gene, can be used, or a competitive binding assaycan be employed.

Labeling and conjugation techniques are known by those skilled in theart and can be used in various nucleic acid and amino acid assays.Methods for producing labeled hybridization or PCR probes for detectingsequences related to nucleic acid sequences encoding a protein, such as,but not limited to, p27, include, but are not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using alabeled nucleotide. Alternatively, nucleic acid sequences encoding apolypeptide encoded by a gene, such as a p27 gene, can be cloned into avector for the production of an mRNA probe. Such vectors are known inthe art, are commercially available, and can be used to synthesize RNAprobes in vitro by addition of labeled nucleotides and an appropriateRNA polymerase such as T7, T3, or SP6. These procedures can be conductedusing a variety of commercially available kits (Amersham PharmaciaBiotech, Promega, and US Biochemical). Suitable reporter molecules orlabels which can be used for ease of detection include radionuclides,enzymes, and fluorescent, chemiluminescent, or chromogenic agents, aswell as substrates, cofactors, inhibitors, and/or magnetic particles.

Expression and Purification of Polypeptides

Host cells transformed with a nucleic acid sequence encoding apolypeptide, such as, but not limited to, p27, can be cultured underconditions suitable for the expression and recovery of the protein fromcell culture. The polypeptide produced by a transformed cell can besecreted or contained intracellularly depending on the sequence and/orthe vector used. Expression vectors containing a nucleic acid sequenceencoding a polypeptide, such as, but not limited to, p27, can bedesigned to contain signal sequences which direct secretion of solublepolypeptide molecules encoded by a gene, such as, but not limited to,p27, or a variant thereof, through a prokaryotic or eukaryotic cellmembrane or which direct the membrane insertion of membrane-bound apolypeptide molecule encoded by a p27 gene or a variant thereof.

Other constructions can also be used to join a gene sequence encoding apolypeptide to a nucleotide sequence encoding a polypeptide domain whichwill facilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).Including cleavable linker sequences (i.e., those specific for Factor Xaor enterokinase (Invitrogen, San Diego, Calif.)) between thepurification domain and a polypeptide encoded by a p27 gene, forexample, also can be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containinga polypeptide encoded by a p27 gene, for example, and 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification by immobilized metal ionaffinity chromatography, while the enterokinase cleavage site provides ameans for purifying the polypeptide encoded by a p27 gene.

A p27 polypeptide can be purified from any human or non-human cell whichexpresses the polypeptide, including those which have been transfectedwith expression constructs that express a p27 protein. A purified p27protein can be separated from other compounds which normally associatewith a protein encoded by a p27 gene in the cell, such as certainproteins, carbohydrates, or lipids, using methods practiced in the art.Non-limiting methods include size exclusion chromatography, ammoniumsulfate fractionation, ion exchange chromatography, affinitychromatography, and preparative gel electrophoresis.

Chemical Synthesis

Nucleic acid sequences comprising a gene, such as, but not limited to, ap27 gene, that encodes a polypeptide can be synthesized, in whole or inpart, using chemical methods known in the art. Alternatively, apolypeptide, such as, but not limited to, p27, can be produced usingchemical methods to synthesize its amino acid sequence, such as bydirect peptide synthesis using solid-phase techniques. Protein synthesiscan either be performed using manual techniques or by automation.Automated synthesis can be achieved, for example, using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally,fragments of p27 polypeptides can be separately synthesized and combinedusing chemical methods to produce a full-length molecule. In oneembodiment, a fragment of a nucleic acid sequence that comprises a p27gene can encompass any portion of at least about 8 consecutivenucleotides of SEQ ID NO: 4. In one embodiment, the fragment cancomprise at least about 10 nucleotides, at least about 15 nucleotides,at least about 20 nucleotides, or at least about 30 nucleotides of SEQID NO: 4. Fragments include all possible nucleotide lengths betweenabout 8 and about 100 nucleotides, for example, lengths between about 15and about 100 nucleotides, or between about 20 and about 100nucleotides.

Other nucleic acid sequences, such as, but not limited to, microRNAtarget sequences, can be synthesized, in whole or in part, usingchemical methods known in the art. Automated synthesis can be achieved,for example, using Applied Biosystems 431 A Peptide Synthesizer (PerkinElmer). In one embodiment, a fragment of a nucleic acid sequence thatcomprises microRNA target sequences can encompass any portion of atleast about 4 consecutive nucleotides of SEQ ID NO: 2, or any nucleicacid sequence complementary to SEQ ID NO. 2. In one embodiment, thefragment can comprise at least about 6 nucleotides, at least about 8nucleotides, at least about 10 nucleotides, or at least about 12nucleotides, at least about 14 nucleotides, at least about 16nucleotides of SEQ ID NO: 2, or any nucleic acid sequence complementaryto SEQ ID NO. 2. Fragments include all possible nucleotide lengthsbetween about 4 and about 40 nucleotides, for example, lengths betweenabout 10 and about 30 nucleotides, or between about 10 and about 20nucleotides.

In another embodiment, a fragment of a nucleic acid sequence thatcomprises microRNA target sequences can encompass one or more nucleicacid sequences of SEQ ID NO: 2, or any nucleic acid sequencecomplementary to SEQ ID NO. 2. In one embodiment, a fragment of anucleic acid sequence that comprises microRNA target sequencesencompasses one nucleic acid sequence of SEQ ID NO: 2, or any nucleicacid sequence complementary to SEQ ID NO. 2. In another embodiment, afragment of a nucleic acid sequence that comprises microRNA targetsequences encompasses two nucleic acid sequence of SEQ ID NO: 2, or anynucleic acid sequence complementary to SEQ ID NO. 2. In one embodiment,a fragment of a nucleic acid sequence that comprises microRNA targetsequences encompasses three nucleic acid sequence of SEQ ID NO: 2, orany nucleic acid sequence complementary to SEQ ID NO. 2. In anotherembodiment, a fragment of a nucleic acid sequence that comprisesmicroRNA target sequences encompasses four nucleic acid sequence of SEQID NO: 2, or any nucleic acid sequence complementary to SEQ ID NO. 2. Inother embodiments, a fragment of a nucleic acid sequence that comprisesmicroRNA target sequences encompasses 5, 6, 7, 8, 9, 10, or more nucleicacid sequences of SEQ ID NO: 2, or any nucleic acid sequencecomplementary to SEQ ID NO. 2.

A synthetic peptide can be substantially purified via high performanceliquid chromatography (HPLC). The composition of a synthetic polypeptideof can be confirmed by amino acid analysis or sequencing. Additionally,any portion of an amino acid sequence comprising a protein encoded by agene can be altered during direct synthesis and/or combined usingchemical methods with sequences from other proteins to produce a variantpolypeptide or a fusion protein.

Pharmaceutical Compositions

In another aspect, the present invention provides for a compositioncomprising a nucleic acid vector, wherein the nucleic acid vectorcomprises a gene of interest and one or more target sequences for amicroRNA within the 3′ UTR region of the gene of interest. In oneembodiment, the composition is a pharmaceutical composition.

In one embodiment, the gene of interest is a human gene. In anotherembodiment, the gene of interest is a non-human gene. In one embodiment,the gene of interest is a p27 gene. In another embodiment, the gene ofinterest is p53. In other embodiments, the gene of interest is a tumorsuppressor gene. A tumor suppressor gene can include, but is not limitedto, APC, RB1, INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4.

In one embodiment, the microRNA is miR-126. In another embodiment, themicro-RNA is miR-143. In another embodiment, the micro-RNA is miR-145.In further embodiments, the microRNA is Let7-f, miR-27b, miR-130a,miR-221, miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a,miR-378, miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the one or more target sequences comprise SEQ IDNO:2. In another embodiment, the one or more target sequences do notcomprise SEQ ID NO:2. In another embodiment, the one or more targetsequences comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the one or more target sequences do not comprisea nucleic acid sequence complementary to SEQ ID NO:2.

In one embodiment, the vector comprises four target sequences for amicroRNA. In another embodiment, the vector comprises three targetsequences. In another embodiment, the vector comprises four targetsequences. In another embodiment, the vector comprises five targetsequences. In another embodiment, the vector comprises six targetsequences. In another embodiment, the vector comprises seven targetsequences. In another embodiment, the vector comprises eight targetsequences. In other embodiments, the vector comprises nine, ten, eleven,twelve, thirteen, fourteen, fifteen, or more target sequences.

In one embodiment, the target sequences for a microRNA are identical. Inanother embodiment, the target sequences for a microRNA are notidentical. The nucleic acid vectors can comprise different combinationsof target sequences for various microRNAs, including, but not limitedto, miR-126, miR-143, miR-145, Let7-f, miR-27b, miR-130a, miR-221,miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a, miR-378,miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the target sequences for a microRNA comprise SEQ IDNO:2. In another embodiment, the target sequences for a microRNA do notcomprise SEQ ID NO:2. In another embodiment, the target sequences for amicroRNA comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the target sequences for a microRNA do notcomprise a nucleic acid sequence complementary to SEQ ID NO:2.

In another embodiment, the vector comprises a second gene of interest.In one embodiment, the second gene of interest is a human gene. Inanother embodiment, the second gene of interest is a non-human gene. Inone embodiment, the second gene of interest is p53. In otherembodiments, the second gene of interest is a tumor suppressor gene. Atumor suppressor gene can include, but is not limited to, APC, RB1,INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4. In one embodiment, the secondgene of interest is an antithrombotic gene. In another embodiment, thesecond gene of interest is an anti-inflammatory gene. In anotherembodiment, the second gene of interest is ENTPD1, TFPI or PTGIS.

In another embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In one embodiment, the vector comprises one target sequence. In anotherembodiment, the vector comprises two target sequences. In anotherembodiment, the vector comprises three target sequences. In anotherembodiment, the vector comprises four target sequences. In anotherembodiment, the vector comprises five target sequences. In anotherembodiment, the vector comprises six target sequences. In anotherembodiment, the vector comprises seven target sequences. In anotherembodiment, the vector comprises eight target sequences. In otherembodiments, the vector comprises nine, ten, eleven, twelve, thirteen,fourteen, fifteen, or more target sequences.

In one embodiment, the nucleic acid vector is a viral vector. In oneembodiment, the viral vector is an adenoviral vector. In anotherembodiment, the viral vector is a lentiviral vector. In one embodiment,the vector is a retroviral vector. In another embodiment, is anoncoviral vector. Examples of adenoviral vectors include vectors derivedfrom adenoviruses such as, but not limited to, adenovirus type 2 (Ad2),adenovirus type 5 (Ad5), adenovirus type 7 (Ad7) and adenovirus type 12(Ad12). In a further embodiment, the vector is an adeno-associatedvector. Examples of adeno-associated vector includes vectors derivedfrom adeno-associated viruses such as, but not limited to,adeno-associated virus type 1 (AAV1), adeno-associated virus type 2(AAV2), adeno-associated virus type 4 (AAV4), adeno-associated virustype 5 (AAV5), adeno-associated virus type 6 (AAV6), adeno-associatedvirus type 7 (AAV7), and adeno-associated virus type 2 (AAV2). Examplesof lentiviral vectors include vectors derived from lentiviruses such as,but not limited to, HIV-1 and HIV-2. Examples of retroviral vectorsinclude vectors derived from retroviruses such as, but not limited to,Moloney murine leukemia virus (MMLV). Examples of oncoviral vectorsinclude vectors derived from oncoviruses such as, but not limited to,Murine Leukemia Virus (MLV), Spleen Necrosis Virus (SNV), Rous sarcomavirus (RSV) and Avian Leukosis Virus (ALV).

Methods of Treatment

In yet another aspect, the invention provides a method of treating orpreventing a cardiovascular disease in a subject in need thereof, themethod comprising administering a nucleic acid vector to the subject,wherein the nucleic acid vector comprises a gene of interest and one ormore target sequences for a microRNA within the 3′ UTR region of thegene of interest.

In one embodiment, the gene of interest is a human gene. In anotherembodiment, the gene of interest is a non-human gene. In one embodiment,the gene of interest is a p27 gene. In another embodiment, the gene ofinterest is p53. In other embodiments, the gene of interest is a tumorsuppressor gene. A tumor suppressor gene can include, but is not limitedto, APC, RB1, INK4, PTEN, MADR2, BRAC1, BRAC2, or DPC4.

In one embodiment, the microRNA is miR-126. In another embodiment, themicro-RNA is miR-143. In another embodiment, the micro-RNA is miR-145.In further embodiments, the microRNA is Let7-f, miR-27b, miR-130a,miR-221, miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a,miR-378, miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the one or more target sequences comprise SEQ IDNO:2. In another embodiment, the one or more target sequences do notcomprise SEQ ID NO:2. In another embodiment, the one or more targetsequences comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the one or more target sequences do not comprisea nucleic acid sequence complementary to SEQ ID NO:2.

In one embodiment, the vector comprises four target sequences for amicroRNA. In another embodiment, the vector comprises three targetsequences. In another embodiment, the vector comprises four targetsequences. In another embodiment, the vector comprises five targetsequences. In another embodiment, the vector comprises six targetsequences. In another embodiment, the vector comprises seven targetsequences. In another embodiment, the vector comprises eight targetsequences. In other embodiments, the vector comprises nine, ten, eleven,twelve, thirteen, fourteen, fifteen, or more target sequences.

In one embodiment, the target sequences for a microRNA are identical. Inanother embodiment, the target sequences for a microRNA are notidentical. The nucleic acid vectors can comprise different combinationsof target sequences for various microRNAs, including, but not limitedto, miR-126, miR-143, miR-145, Let7-f, miR-27b, miR-130a, miR-221,miR-222, miR-17, miR-18a, miR-19a, miR-20a, miR-19b, miR-92a, miR-378,miR-210, miR-15, miR-16, miR-20b, miR-155, or miR-21.

In one embodiment, the target sequences for a microRNA comprise SEQ IDNO:2. In another embodiment, the target sequences for a microRNA do notcomprise SEQ ID NO:2. In another embodiment, the target sequences for amicroRNA comprise a nucleic acid sequence complementary to SEQ ID NO:2.In another embodiment, the target sequences for a microRNA do notcomprise a nucleic acid sequence complementary to SEQ ID NO:2.

In another embodiment, the vector comprises a second gene of interest.In one embodiment, the second gene of interest is a human gene. Inanother embodiment, the second gene of interest is a non-human gene. Inone embodiment, the second gene of interest is p53. In otherembodiments, the second gene of interest is a tumor suppressor gene. Atumor suppressor gene can include, but is not limited to, APC, RB1,INK4, PTEN, MADR2, BRAC1. BRAC2, or DPC4. In one embodiment, the secondgene of interest is an antithrombotic gene. In another embodiment, thesecond gene of interest is an anti-inflammatory gene. In anotherembodiment, the second gene of interest is ENTPD1, TFPI or PTGIS.

In another embodiment, the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.In one embodiment, the vector comprises one target sequence. In anotherembodiment, the vector comprises two target sequences. In anotherembodiment, the vector comprises three target sequences. In anotherembodiment, the vector comprises four target sequences. In anotherembodiment, the vector comprises five target sequences. In anotherembodiment, the vector comprises six target sequences. In anotherembodiment, the vector comprises seven target sequences. In anotherembodiment, the vector comprises eight target sequences. In otherembodiments, the vector comprises nine, ten, eleven, twelve, thirteen,fourteen, fifteen, or more target sequences.

In one embodiment, the nucleic acid vector is a viral vector. In oneembodiment, the viral vector is an adenoviral vector. In anotherembodiment, the viral vector is a lentiviral vector. In one embodiment,the vector is a retroviral vector. In another embodiment, is anoncoviral vector. Examples of adenoviral vectors include vectors derivedfrom adenoviruses such as, but not limited to, adenovirus type 2 (Ad2),adenovirus type 5 (Ad5), adenovirus type 7 (Ad7) and adenovirus type 12(Ad12). In a further embodiment, the vector is an adeno-associatedvector. Examples of adeno-associated vector includes vectors derivedfrom adeno-associated viruses such as, but not limited to,adeno-associated virus type 1 (AAV1), adeno-associated virus type 2(AAV2), adeno-associated virus type 4 (AAV4), adeno-associated virustype 5 (AAV5), adeno-associated virus type 6 (AAV6), adeno-associatedvirus type 7 (AAV7), and adeno-associated virus type 2 (AAV2). Examplesof lentiviral vectors include vectors derived from lentiviruses such as,but not limited to, HIV-1 and HIV-2. Examples of retroviral vectorsinclude vectors derived from retroviruses such as, but not limited to,Moloney murine leukemia virus (MMLV). Examples of oncoviral vectorsinclude vectors derived from oncoviruses such as, but not limited to,Murine Leukemia Virus (MLV), Spleen Necrosis Virus (SNV), Rous sarcomavirus (RSV) and Avian Leukosis Virus (ALV).

In one embodiment, the nucleic acid vector is delivered into a cell ofthe subject. Delivery can be conducted by any method known to one ofskill in the art, including, but not limited to, injection,transfection, lipofection, microinjection, calcium phosphate or calciumchloride precipitation, DEAE-dextrin-mediated transfection, orelectroporation. Electroporation is carried out at approximate voltageand capacitance to result in entry of the DNA construct(s) into cells ofinterest. Other methods used to transfect cells can also includemodified calcium phosphate precipitation, polybrene precipitation,liposome fusion, and receptor-mediated gene delivery.

In another embodiment, the cell expresses the microRNA. In anotherembodiment, the cell does not express the microRNA.

In one embodiment, the gene of interest is not expressed in the cell. Inanother embodiment, the gene of interest is expressed in the cell. Inone embodiment, the second gene of interest is not expressed in thecell. In another embodiment, the second gene of interest is expressed inthe cell.

In one embodiment, the cell is an endothelial cell. In anotherembodiment, the cell is a vascular smooth muscle cell. In furtherembodiments, the cell may be from the germ line or somatic, totipotentor pluripotent, dividing or non-dividing, parenchyma or epithelium,immortalized or transformed, or the like. The cell may be a stem cell ora differentiated cell. Cell types that are differentiated include, butare not limited to, adipocytes, fibroblasts, myocytes, cardiomyocytes,endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes,macrophages, neutrophils, eosinophils, basophils, mast cells,leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts,osteoclasts, hepatocytes, and cells of the endocrine and exocrineglands.

In another embodiment, the cell is a cancer cell. The cancer can be, butis not limited to, breast cancer, lung cancer, kidney cancer, braincancer, liver cancer, colorectal cancers, progressive lungadenocarcinoma, lymphomas, leukemias, adenocarcinomas and sarcomas.

In one embodiment, the cardiovascular disease is coronary arterydisease. In another embodiment, the cardiovascular disease isatherosclerotic coronary artery disease. In further embodiments, thecardiovascular disease includes, but is not limited to, coronaryvasospasm, restenosis, myocardial ischemia, stent induced injury, stentthrombosis, cardiomyopathy, hypertensive heart disease, heart failure,cor pulmonale, cardiac dysrhythmias, inflammatory heart disease,valvular heart disease, peripheral arterial disease, cerebrovasculardisease, congenital heart disease, atherosclerosis, arterial injury andrheumatic heart disease. The methods of the invention can also be usedfor other vascular conditions, such as vascular access failure, forexample, in hemodialysis.

Pharmaceutical Compositions and Administration for Therapy

Nucleic acid vectors of the invention can be administered to the subjectonce (e.g., as a single injection or deposition). Alternatively, nucleicacid vectors of the invention can be administered once or twice daily toa subject in need thereof for a period of from about two to abouttwenty-eight days, or from about seven to about ten days. Nucleic acidvectors of the invention can also be administered once or twice daily toa subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 timesper year, or a combination thereof. Furthermore, nucleic acid vectors ofthe invention can be co-administrated with another therapeutic. Where adosage regimen comprises multiple administrations, the effective amountof the nucleic acid vectors administered to the subject can comprise thetotal amount of the nucleic acid vectors administered over the entiredosage regimen.

Nucleic acid vectors of the invention can be administered to a subjectby any means suitable for delivering the nucleic acid vectors to cellsof the subject. For example, nucleic acid vectors can be administered bymethods suitable to transfect cells. Transfection methods for eukaryoticcells are well known in the art, and include direct injection of anucleic acid into the nucleus or pronucleus of a cell; electroporation;liposome transfer or transfer mediated by lipophilic materials; receptormediated nucleic acid delivery, bioballistic or particle acceleration;calcium phosphate precipitation, and transfection mediated by viralvectors.

The nucleic acid vectors of the invention may be administered to asubject in an amount effective to treat or prevent a cardiovasculardisease, such as, but not limited to, coronary artery disease. One ofskill in the art can readily determine what will be an effective amountof the nucleic acid vectors of the invention to be administered to asubject, taking into account whether the nucleic acid vectors are beingused prophylactically or therapeutically, and taking into account otherfactors such as the age, weight and sex of the subject, any other drugsthat the subject may be taking, any allergies or contraindications thatthe subject may have, and the like. For example, an effective amount canbe determined by the skilled artisan using known procedures, includinganalysis of titration curves established in vitro or in vivo. Also, oneof skill in the art can determine the effective dose from performingpilot experiments in suitable animal model species and scaling the dosesup or down depending on the subjects weight etc. Effective amounts canalso be determined by performing clinical trials in individuals of thesame species as the subject, for example starting at a low dose andgradually increasing the dose and monitoring the effects on a metabolicdisorder, or coronary artery disease. Appropriate dosing regimens canalso be determined by one of skill in the art without undueexperimentation, in order to determine, for example, whether toadminister the agent in one single dose or in multiple doses, and in thecase of multiple doses, to determine an effective interval betweendoses.

A therapeutically effective dose of a nucleic acid vector of theinvention that treats or prevents a cardiovascular disease, such as, butnot limited to, coronary artery disease, can depend upon a number offactors known to those of ordinary skill in the art. The dose(s) of thenucleic acid vectors can vary, for example, depending upon the identity,size, and condition of the subject or sample being treated, furtherdepending upon the route by which the composition comprising nucleicacid vectors is to be administered, if applicable, and the effect whichthe practitioner desires the nucleic acid vectors to have upon thetarget of interest. These amounts can be readily determined by a skilledartisan. Any of the therapeutic applications described herein can beapplied to any subject in need of such therapy, including, for example,a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, apig, a sheep, a goat, or a human.

The compositions of this invention can be formulated and administered toreduce the symptoms associated with a cardiovascular disease, such as,but not limited to, coronary artery disease, by any means that producescontact of the active ingredient with the agent's site of action in thebody of a subject, such as a human or animal (e.g., a dog, cat, orhorse). They can be administered by any conventional means available foruse in conjunction with pharmaceuticals, either as individualtherapeutic active ingredients or in a combination of therapeutic activeingredients. They can be administered alone, but are generallyadministered with a pharmaceutical carrier selected on the basis of thechosen route of administration and standard pharmaceutical practice.

Pharmaceutical compositions for use in accordance with the invention canbe formulated in conventional manner using one or more physiologicallyacceptable carriers or excipients. The therapeutic compositions of theinvention can be formulated for a variety of routes of administration,including systemic or localized administration. Techniques andformulations generally can be found in Remmington's PharmaceuticalSciences, Meade Publishing Co., Easton, Pa. (20^(th) Ed., 2000), theentire disclosure of which is herein incorporated by reference. Forsystemic administration, an injection is useful, includingintramuscular, intravenous, intraperitoneal, and subcutaneous. Forinjection, the therapeutic compositions of the invention can beformulated in liquid solutions, for example in physiologicallycompatible buffers such as Hank's solution or Ringer's solution. Inaddition, the therapeutic compositions can be formulated in solid formand redissolved or suspended immediately prior to use. Lyophilized formsare also included. Pharmaceutical compositions of the present inventionare characterized as being at least sterile and pyrogen-free. Thesepharmaceutical formulations include formulations for human andveterinary use.

According to the invention, a pharmaceutically acceptable carrier cancomprise any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Any conventional media or agent that is compatible with theactive ingredient can be used. Supplementary active compounds can alsobe incorporated into the compositions.

The invention also provides for a kit that comprises a pharmaceuticallyacceptable carrier and one or more nucleic acid vector(s) of theinvention packaged with instructions for use.

A pharmaceutical composition containing nucleic acid vectors of theinvention can be administered in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed herein.The compositions can be administered alone or in combination with atleast one other agent, such as a stabilizing compound, which can beadministered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions can be administered to a patient alone, or incombination with other agents, drugs or hormones.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, a pharmaceutically acceptable polyol like glycerol,propylene glycol, liquid polyetheylene glycol, and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it can be useful to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of injectable compositions can bebrought about by incorporating an agent which delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating thenucleic acid vectors of the invention in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedherein, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the nucleic acid vectors intoa sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated herein. In the case ofsterile powders for the preparation of sterile injectable solutions,examples of useful preparation methods are vacuum drying andfreeze-drying which yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the nucleic acid vectors are formulated intoointments, salves, gels, or creams as generally known in the art. Insome embodiments, the nucleic acid vectors can be applied viatransdermal delivery systems, which slowly releases the nucleic acidvectors for percutaneous absorption. Permeation enhancers can be used tofacilitate transdermal penetration of the active factors in theconditioned media. Transdermal patches are described in for example,U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No.5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat.No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S.Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110;and U.S. Pat. No. 4,921,475.

Administration of the nucleic acid vectors is not restricted to a singleroute, but may encompass administration by multiple routes. Forinstance, exemplary administrations by multiple routes include, amongothers, a combination of intradermal and intramuscular administration,or intradermal and subcutaneous administration. Multiple administrationsmay be sequential or concurrent. Other modes of application by multipleroutes will be apparent to the skilled artisan.

The nucleic acid vectors of the invention may be formulated intocompositions for administration to subjects for the treatment and/orprevention of a cardiovascular disease, such as, but not limited to,coronary artery disease. Such compositions may comprise the nucleic acidvectors of the invention in admixture with one or more pharmaceuticallyacceptable diluents and/or carriers and optionally one or more otherpharmaceutically acceptable additives. The pharmaceutically-acceptablediluents and/or carriers and any other additives must be “acceptable” inthe sense of being compatible with the other ingredients of thecomposition and not deleterious to the subject to whom the compositionwill be administered. One of skill in the art can readily formulate thenucleic acid vectors of the invention into compositions suitable foradministration to subjects, such as human subjects, for example usingthe teaching a standard text such as Remington's PharmaceuticalSciences, 18th ed, (Mack Publishing Company: Easton, Pa., 1990), pp.1635-36), and by taking into account the selected route of delivery.

Examples of diluents and/or carriers and/or other additives that may beused include, but are not limited to, water, glycols, oils, alcohols,aqueous solvents, organic solvents, DMSO, saline solutions,physiological buffer solutions, peptide carriers, starches, sugars,preservatives, antioxidants, coloring agents, pH buffering agents,granulating agents, lubricants, binders, disintegrating agents,emulsifiers, binders, excipients, extenders, glidants, solubilizers,stabilizers, surface active agents, suspending agents, tonicity agents,viscosity-altering agents, carboxymethyl cellulose, crystallinecellulose, glycerin, gum arabic, lactose, magnesium stearate, methylcellulose, powders, saline, sodium alginate. The combination of diluentsand/or carriers and/or other additives used can be varied taking intoaccount the nature of the active agents used (for example the solubilityand stability of the active agents), the route of delivery (e.g. oral,parenteral, etc.), whether the agents are to be delivered over anextended period (such as from a controlled-release capsule), whether theagents are to be co-administered with other agents, and various otherfactors. One of skill in the art will readily be able to formulate thenucleic acid vectors for the desired use without undue experimentation.

The compositions of the invention may be administered to a subject byany suitable method that allows the agent to exert its effect on thesubject in vivo. For example, the compositions may be administered tothe subject by known procedures including, but not limited to, by oraladministration, sublingual or buccal administration, parenteraladministration, transdermal administration, via inhalation, via nasaldelivery, vaginally, rectally, and intramuscularly. The compositions ofthe invention may be administered parenterally, or by epifascial,intracapsular, intracutaneous, subcutaneous, intradermal, intrathecal,intramuscular, intraperitoneal, intrasternal, intravascular,intravenous, parenchymatous, or sublingual delivery. Delivery may be byinjection, infusion, catheter delivery, or some other means, such as bytablet or spray. In one embodiment, the nucleic acid vectors of theinvention are administered to the subject by way of delivery directly tothe heart tissue, such as by way of a catheter inserted into, or in theproximity of the subject's heart, or by using delivery vehicles capableof targeting the drug to the heart. For example, the nucleic acidvectors of the invention may be conjugated to or administered inconjunction with an agent that is targeted to the heart, such as anantibody or antibody fragment. In one embodiment, the nucleic acidvectors of the invention are administered to the subject by way ofdelivery directly to the tissue of interest, such as by way of acatheter inserted into, or in the proximity of the subject's tissue ofinterest, or by using delivery vehicles capable of targeting the nucleicacid vectors to the muscle, such as an antibody or antibody fragment.

For oral administration, a formulation of the nucleic acid vectors ofthe invention may be presented as capsules, tablets, powders, granules,or as a suspension or solution. The formulation may contain conventionaladditives, such as lactose, mannitol, cornstarch or potato starch,binders, crystalline cellulose, cellulose derivatives, acacia,cornstarch, gelatins, disintegrators, potato starch, sodiumcarboxymethylcellulose, dibasic calcium phosphate, anhydrous or sodiumstarch glycolate, lubricants, and/or or magnesium stearate.

For parenteral administration (i.e., administration by through a routeother than the alimentary canal), the nucleic acid vectors of theinvention may be combined with a sterile aqueous solution that isisotonic with the blood of the subject. Such a formulation may beprepared by dissolving the active ingredient in water containingphysiologically-compatible substances, such as sodium chloride, glycineand the like, and having a buffered pH compatible with physiologicalconditions, so as to produce an aqueous solution, then rendering thesolution sterile. The formulation may be presented in unit or multi-dosecontainers, such as sealed ampoules or vials. The formulation may bedelivered by injection, infusion, or other means known in the art.

For transdermal administration, the nucleic acid vectors of theinvention may be combined with skin penetration enhancers, such aspropylene glycol, polyethylene glycol, isopropanol, ethanol, oleic acid,N-methylpyrrolidone and the like, which increase the permeability of theskin to the nucleic acid vectors of the invention and permit the nucleicacid vectors to penetrate through the skin and into the bloodstream. Thenucleic acid vectors of the invention also may be further combined witha polymeric substance, such as ethylcellulose, hydroxypropyl cellulose,ethylene/vinylacetate, polyvinyl pyrrolidone, and the like, to providethe composition in gel form, which are dissolved in a solvent, such asmethylene chloride, evaporated to the desired viscosity and then appliedto backing material to provide a patch.

In some embodiments, the nucleic acid vectors of the invention areprovided in unit dose form such as a tablet, capsule or single-doseinjection or infusion vial.

Combination Therapy

According to the methods of the invention, a nucleic acid vector of theinvention can be administered to a subject either as a single agent, orin combination with one or more other agents. In one embodiment, anucleic acid vector of the invention is administered to a subject as asingle agent. In one embodiment, a nucleic acid vector of the inventionis administered to a subject alone. In one embodiment, a nucleic acidvector of the invention is administered to a subject in combination withone or more other agents.

In certain embodiments, a nucleic acid vector of the invention may beused in combination with other agents that are used for the treatment orprevention of a cardiovascular disease, such as, but not limited to,coronary artery disease in a subject. In certain embodiments, a nucleicacid vector of the invention may be used in combination with otheragents that are not used for the treatment or prevention of acardiovascular disease, such as, but not limited to, coronary arterydisease in a subject. In one embodiment, a nucleic acid vector of theinvention may be delivered to a subject as part of the samepharmaceutical composition or formulation containing one or moreadditional active agents. In another embodiment, a nucleic acid vectorof the invention may be delivered to a subject in a composition orformulation containing only that active agent, while one or more otheragents are administered to the subject in one or more separatecompositions or formulations. In one embodiment, the other agents arenot used for the treatment or prevention of a cardiovascular disease,such as, but not limited to, coronary artery disease in a subject. Inanother embodiment, the other agents are used for the treatment orprevention of a cardiovascular disease, such as, but not limited to,coronary artery disease in a subject.

A nucleic acid vector of the invention and the other agents that areused for the treatment or prevention of a cardiovascular disease, suchas, but not limited to, coronary artery disease in a subject, may beadministered to the subject at the same time, or at different times. Anucleic acid vector of the invention and the other agents that are notused for the treatment or prevention of a cardiovascular disease, suchas, but not limited to, coronary artery disease, may be administered tothe subject at the same time, or at different times. For example, anucleic acid vector of the invention and the other agents may beadministered within minutes, hours, days, weeks, or months of eachother, for example as part of the overall treatment regimen of asubject. In some embodiments, a nucleic acid vector of the invention maybe administered prior to the administration of other agents. In otherembodiments, a nucleic acid vector of the invention may be administeredsubsequent to the administration of other agents.

In some embodiments, the administration of a nucleic acid vector of theinvention in combination with one or more other agents has an additiveeffect, in comparison with administration of the nucleic acid vector ofthe invention alone, or administration of the one or more other agentsalone. In other embodiments, the administration of a nucleic acid vectorof the invention in combination with one or more other agents has asynergistic effect, in comparison with administration of the nucleicacid vector of the invention alone, or administration of the one or moreother agents alone. In some embodiments, the administration of a nucleicacid vector of the invention in combination with one or more otheragents can help reduce side effects, in comparison with administrationof the nucleic acid vector of the invention alone, or administration ofthe one or more other agents alone.

In some embodiments, the nucleic acid vector of the invention is used asan adjuvant therapy. In other embodiments, the nucleic acid vector ofthe invention is used in combination with an adjuvant therapy.

The invention may also be used in combination with known therapies for acardiovascular disease. Examples include, but are not limited to,aspirin; statins, such as atorvastatin (Lipitor, Torvast), fluvastatin(Lescol), lovastatin (Mevacor, Altocor, Altoprev), pitavastatin (Livalo,Pitava), pravastatin (Pravachol, Selektine, Lipostat), rosuvastatin(Crestor), and simvastatin (Zocor, Lipex); nitroglycerin;angiotensin-converting enzyme (ACE) inhibitors, such as enalapril(Vasotec®), lisinopril (Zestril®, Prinvil®), ramipril (Altace®) andcaptopril (Capoten®); calcium channel blockers, such as verapamil(Calan®, Isoptin®); and beta-blockers, such as carvedilol (Cored®) andmetoprolol (Lopressor®, Toprol XL®).

The invention may also be used in combination with surgical or otherinterventional treatment regimens used for treatment of a cardiovasculardisease, such as, but not limited to, use of a device, including, butnot limited to, a stent, a rapamycin-coated stent, a drug-eluting stent,a bare-metal stent, a pacemaker, an implantablecardioverter-defibrillator (ICD) or a ventricular assist device (VAD).

Subjects

According to the methods of the invention, the subject or patient can beany animal that has or is diagnosed with a cardiovascular disease, suchas, but not limited to, coronary artery disease. According to themethods of the invention, the subject or patient can be any animal thatis predisposed to or is at risk of developing a cardiovascular disease,such as, but not limited to, coronary artery disease. In preferredembodiments, the subject is a human subject. In some embodiments, thesubject is a rodent, such as a mouse. In some embodiments, the subjectis a cow, pig, sheep, goat, cat, horse, dog, and/or any other species ofanimal used as livestock or kept as pets.

In some embodiments, the subject is already suspected to have acardiovascular disease, such as, but not limited to, coronary arterydisease. In other embodiments, the subject is being treated for acardiovascular disease, such as, but not limited to, coronary arterydisease, before being treated according to the methods of the invention.In other embodiments, the subject is not being treated for acardiovascular disease, such as, but not limited to, coronary arterydisease, before being treated according to the methods of the invention.

EXAMPLES

The following examples illustrate the present invention, and are setforth to aid in the understanding of the invention, and should not beconstrued to limit in any way the scope of the invention as defined inthe statements of the invention which follow thereafter.

The Examples described below are provided to illustrate aspects of thepresent invention and are not included for the purpose of limiting theinvention.

Example 1 MicroRNA-Based Strategy to Selectively Preserve EndothelialFunction

The numbers in parentheses below refer to the corresponding numberedreference(s) at the end of this section.

Coronary artery disease (CAD) is a leading cause of death worldwide.Despite the benefits of drug-eluting stents (DES) concerns have beenraised over their safety due to delayed endothelial cell (EC) coveragewhich promotes vascular restenosis and thrombosis, requiring prolongeddual anti-platelet therapy. Herein is shown a new therapeutic approachexploiting the EC specific microRNA-126 to prevent restenosis whilepreserving EC function.

Percutaneous coronary intervention (PCI) is one of the most commonlyperformed interventions that have transformed the practice ofrevascularization for CAD (1). However, the major drawback of thisprocedure is the proliferation and subsequent accumulation of vascularsmooth muscle cells (VSMC), leading to restenosis (2, 3). This process,also known as intimal hyperplasia, is triggered by the injury of thearterial wall and the concurrent endothelial denudation. The advent ofDES, capable of delivering an inhibitor of cell proliferation in situhas decreased, but not eliminated, the occurrence of restenosis (2, 4).The drugs that elute from the stent not only inhibit VSMC, but also ECproliferation and migration (5, 6), increasing the risk of latethrombosis, a rare and potentially catastrophic event that is caused byincomplete re-endothelization (1, 7). The ideal therapy can provideVSMC-selective anti-proliferative activity without affecting EC.

Recently, one of the crucial breakthroughs in the study of generegulation has been the discovery of microRNAs (miRs), a class ofendogenous small non-coding RNA (8, 9), miRs regulate the expression ofmuch of the transcriptome via degradation of their target mRNA and/orinhibiting translation, miR-126 is expressed in a cell-specific manner(8, 10) that enabled to selectively inhibit VSMC proliferation whilepreserving the capability of EC to proliferate and migrate in order tore-endothelialize the vessel. Since the expression level of miR-126is >600-fold higher in EC than in VSMC (FIG. 2A), this miR was selectedto design a VSMC-specific therapy.

The cyclin-dependent kinase inhibitor p27^(KIP1) (p27) is known to be apotent inhibitor of cell proliferation (3). Different adenoviral (Ad)vectors were designed to overexpress p27 (FIGS. 4A-C): a CMV-GFP-p27(Ad-p27) and a CMV-GFP-p27 containing 4 target sequences of miR-126 inits 3′UTR (Ad-p27-126TS) to selectively avoid overexpression of p27 inEC. A CMV-GFP virus (Ad-GFP) was used as control. EC and VSMC wereinfected with these Ad and the expressions of GFP and p27 were assessedby immunoblotting (FIGS. 2B,C). p27 was not overexpressed when Ad-GFPwas used in both VSMC and EC, while p27 was highly overexpressed in bothcell types when Ad-p27 was used. After Ad-p27-126TS infection, p27 wasoverexpressed in VSMC, which do not express mir-126. Since EC do expressmiR-126, which binds to the four target sequences in p27-3′UTR, p27overexpression was abolished (FIGS. 2B,C). Next, the effect of the threeAd vectors on the proliferation (FIG. 2D) and migration (FIGS. 2E-H) ofEC and VSMC was tested. Ad-p27 inhibited proliferation andmigration >70% in both cell types (FIGS. 2D-H). Importantly, VSMCinfected with Ad-p27-126TS still showed >70% inhibition of proliferationand migration, while EC were not affected (FIGS. 2D-H). In addition, ECnetwork formation was examined. EC infected with Ad-p27, whichoverexpress p27, displayed a significant decrease in the network-likeformation, whereas Ad-p27-126TS infected EC formed networks comparableto Ad-GFP infected cells (FIGS. 2I,J).

To test this approach in vivo, balloon injury of the rat carotid arterywas used (11) and the injured vessel was infected with the three Ad. Twoweeks after the injury, the efficiency of the infection (GFPimmunostaining. FIG. 5A), the neointimal formation (FIG. 5B and FIGS.4A,B) and the integrity of the endothelium (FIGS. 3A, C) were evaluated.Since pathological and clinical studies have reported incompleteneointimal coverage after PCI leading to thrombosis (12, 13),hypercoagulability was assessed by measuring plasma levels of D-dimer(14). The arteries infected with Ad-GFP demonstrated >2-fold increase inthe neointima/media ratio after the balloon injury, and >80% decrease inre-endothelization (FIGS. 3A-C), as well as higher levels of D-dimer(FIG. 3D) compared to animals with uninjured arteries. Ad-p27inhibited >85% of the neointimal formation and re-endothelizationwithout affecting hypercoagulability (FIGS. 3A-D). When Ad-p27-126TS wasused, the inhibition of the neointimal formation was similar to Ad-p27,widespread re-endothelization was observed, and the D-dimer level wasdecreased similarly to the uninjured control (FIGS. 3A-D).

To investigate whether the new endothelium was functional, a vascularreactivity assay was performed on carotid rings harvested from rats 2weeks after vessel surgery (15). Carotid arteries infected with Ad-GFPdisplayed a blunted vasodilation response to acetylcholine, most likelydue to restenosis (FIG. 3E). The Ad-p27-infected vessels also showed animpaired vasodilation response, comparable to denuded control vessels.Arteries infected with Ad-p27-126TS, in contrast, showed a normalvasodilation response, comparable to uninjured control vessels (FIG.3E).

Taken together, the data demonstrate for the first time that byexploiting the EC-specific miR-126, it is possible to specificallyoverexpress p27 in VSMC to inhibit proliferation and migration withoutaffecting EC re-endothelialization. Normal endothelial function, asassessed by the reduction in D-dimer levels andacetylcholine-responsiveness, was also restored. This approach offers abasis for the development of new specific and selective therapeuticstrategies to treat diverse pathological VSMC proliferative conditions,including restenosis, stent thrombosis, transplant vasculopathy, andvein graft failure. This new approach can be safer than thenon-selective DES currently used in the clinical practice and can reducethe need for prolonged dual antiplatelet therapy following PCI. Furtherinvestigations can optimize the gene transfer to apply this approach inthe clinical setting (8).

REFERENCES

-   1. Garg, S. & Serruys, P. W. Journal of the American College of    Cardiology 56, S1-42 (2010).-   2. Jukema, J. W., Verschuren, J. J. W., Ahmed, T. A. N. &    Quax, P. H. Nat. Rev. Cardiol. 9, 53-62 (2012).-   3. Marx, S. O., Totary-Jain, H. & Marks, A. R. Circ Cardiovasc    Interv 4, 104-111 (2011).-   4. Cassese, S. & Kastrati, A. JAMA 308, 814-815 (2012).-   5. Kotani, J., et al. Journal of the American College of Cardiology    47, 2108-2111 (2006).-   6. Liu, H. T., et al. Tex Heart Inst J 37, 194-201 (2010).-   7. Wenaweser. P., et al. Journal of the American College of    Cardiology 52, 1134-1140 (2008).-   8. van Rooij, E. & Olson, E. N. Nat Rev Drug Discov 11, 860-872    (2012).-   9. Brown, B. D., Venneri, M. A., Zingale, A., Sergi Sergi, L. &    Naldini, L. Nat Med 12, 585-591 (2006).-   10. Wang, S., et al. Dev Cell 15, 261-271 (2008).-   11. Iaccarino, G., Smithwick, L. A., Lefkowitz, R. J. & Koch, W. J.    Proc Natl Acad Sci USA 96, 3945-3950 (1999).-   12. Hayashi, S., et al. The American journal of pathology 175,    2226-2234 (2009).-   13. Iakovou, I., et al. JAMA 293, 2126-2130 (2005).-   14. Yamaguchi. K., et al. Int J Cardiol 153, 272-276 (2011).-   15. Santulli, G., el al., J Am Heart Assoc 1, e001081 (2012).

Methods

The numbers in parentheses below refer to the corresponding numberedreference(s) at the end of this section.

Adenovirus Design.

Recombinant adenoviruses containing human p27 (Ad-p27) and p27 thatcontains four tandem targeting sequences for hsa-miR-126-3p introducedin its 3′UTR region (Ad-p27-126TS) were constructed using AdEasy XLAdenoviral Vector System (Agilent Technologies, Santa Clara, Calif.),following the manufacturer's manual. Briefly, p27 was amplified withprimers 5′-agtcggtaccaccATGTCAAACGTGCGAGTGTCTAACGG-3′ and5′-gatctgtacaggatccTTACGTTTGACGTCTTCTGAGGCC-3′ (start and stop codons inbold and restriction sites in lowercase) and subcloned into a carriervector. Complementary oligonucleotides containing four targetingsequences for miR-126 (in bold)(5′-aattcATCGCATTATTACTCACGGTACGAAATCCGCATTATTACTCACGGTACGAAATCCGCATTATTACTCACGGTACGAAATCCGCATTATTACTCA CGGTACGAATg-3′ and5′-aattcATTCGTACCGTGAGTAATAATGCGGATTTCGTACCGTGAGTAATAATGCGGAITCGTACCGTGAGTAATAATGCGGATTTCGTACCGTGAGT AATAATGCGATg-3′) wereannealed and subcloned in the 3′-UTR region of p27 with EcoRI. Next, p27and p27-126TSx4 were amplified from the carrier vector with thefollowing pairs of primers:5′-agtcggtaccaccATGTCAAACGTGCGAGTGTCTAACGG-3′ as a sense primer for bothconstructs, and 5′-agtcctcgagTTACGTTTGACGTCTTCTGAGGCC-3′ and5′-agtcctcgagaTTCGTACCGTGAGTAATAATGCGGATTTC-3′ as antisense primers forp27 and p27-126TSx4, respectively. The resulting PCR products weresubcloned into pAdTrack-CMV shuttle vector (using Acc65 I and Xho Irestriction sites in lowercase) under the control of a cytomegalovirus(CMV) promoter. All the constructs also contain a green fluorescenceprotein (GFP) under the control of a separate CMV promoter. All finalvirus constructs were purified by ultracentrifugation in CsCl gradient,dialyzed, confirmed by sequencing and titrated in HEK cells.

Cell Culture.

Human umbilical venous endothelial cells (HUVEC) were cultured in EGM2Bullet Kit medium (Lonza, Basel, Switzerland) on dishes coated with type1 rat tail collagen (VWR, Radnor, Pa.). Human umbilical vascular smoothmuscle cells (VSMC) and SmGM-2 Bullet Kit medium were purchased fromLonza. The cells in all assays were below passage 6 and cultured at adensity that allowed cell division throughout the course of theexperiment.

Quantitative Real Time-PCR.

TaqMan® microRNA Assays (Life Technologies) were used to quantify maturemiRNAs. cDNA was synthesized by priming with miRNA-specific loopedprimers, or U18 as endogenous control. Total RNA (100 ng) extracted withmiRNeasy Mini Kit (Quiagen, Germantown, Md.) was used for each reversetranscription reaction according to the manufacturer's specificationsand incubated for 30 min at 16° C., 30 min at 42° C., and 5 min at 85°C. and stored at 4° C. PCR was performed using 4.5 ng cDNA, 1× TaqMan®Universal PCR Master Mix (P/N: 4324018) and TaqMan® MicroRNA assay. Allreactions, excluding no-template controls and non-reverse-transcribedcontrols were run in triplicate, incubated in 96-well plates at 95° C.for 20 seconds, followed by 40 cycles of 95° C. for 3 seconds and 60° C.for 30 seconds using the ABI 7500 Fast Real Time PCR Detection System.Real-time PCR data were analyzed using the comparative CT method,normalizing against the expression of U18.

Proliferation, Migration, and Network Formation Assays.

VSMC and EC were infected with 20 or 30 pfu/cell, respectively.Proliferation assay was carried out six days after infection by usingthe Cell-Proliferation-Reagent kit (Roche, Basel, Switzerland) accordingto the manufacturer's instruction. The absorbance was measured at 450 nm(with 690 nm reference wavelength) using Infinite® F500 microplatereader (Tecan, Männedorf, Switzerland). Cellular migration was assessedas previously described (1). The formation of network-like structuresassay was performed and quantified as previously described and validated(1, 2). Several fields of view were captured per well and experimentswere repeated three times by blinded observers.

Immunoblotting.

Immunoblot analysis was performed as previously described (3). Briefly,samples were resolved by SDS-PAGE and proteins transferred topoly-vinylidene difluoride and visualized by immunoblotting usinginfrared-labeled anti-rabbit (red) and anti mouse (green) secondaryantibodies (1:10,000, LI-COR Biosciences. Lincoln, Nebr.). Bandintensities were quantified with the Odyssey Infrared Imaging System(LI-COR Biosciences). Blots were probed with the following antibodies:rabbit anti-GAPDH (Cell signaling Technology®), rabbit anti-GFP (LifeTechnologies) and mouse anti-p27 (BD Transduction Laboratories™), SanJose, Calif.). Data are presented as arbitrary units after normalizationfor GAPDH as loading control.

In Vivo Balloon Injury.

The animals were housed in a 22° C. room with a 12-hour light/darkcycle. Balloon injury of the right carotid artery was performed in maleSprague Dawley rats (weighing 300±30 g, Harlan, South Easton, Mass.)using a HyperGlide™ ballon catheter (Micro Therapeutics, Inc. Irvine,Calif.), partially modifying a previously described procedure (4).Briefly, the animals were anesthetized by isoflurane (4%) inhalation andmaintained by mask ventilation (isoflurane 2%), and the right common,external and internal carotid arteries were exposed and isolated.Through the external carotid, the balloon catheter was introduced in thecommon carotid and inflated 7 times. After injury, the common carotidartery was flushed twice with PBS, and a solution of PBS and adenovirus[5×10⁹ plaque-forming unit (pfu)/100 μl] was injected and allowed toincubate in the common carotid in the absence of flow for 20 min. Duringthis procedure the tension of common carotid artery was maintained byplacing microvascular clips (Harvard Apparatus, Holliston, Mass.) on theinternal and the common carotids (5). The adenovirus then was removed,the external carotid was tied and the blood flow was restored throughthe common and the internal carotid arteries. Following wound closure,the rats were given ad libitum access to food and water. All experimentswere performed by blinded investigators.

D-Dimer Measurement.

Hypercoagulability was assessed measuring plasma levels of D-dimer (6)using a rat immunoassay (USCN Life Science Inc. Houston, Tex.) inaccordance to the manufacturer's instructions.

Morphological Analysis.

Two weeks after surgery, the rats were euthanized and the carotidarteries were fixed by perfusion at 100 mmHg with 100 ml of PBS,followed by 80 ml of PBS containing 4% paraformaldehyde via a cannulaplaced in the left ventricle. Both right and left common carotidarteries were then excised, cut in two portions and embedded in optimalcutting temperature (OCT) medium (Sakura Finetek, Tokyo, Japan) forcryosectioning. Subsequently, 10 μm sections were cut every 20 μm andsubmitted in toto for histological evaluation. Sections were processedfor staining with anti-α-smooth muscle actin (α-SMA, Sigma-Aldrich,1:1000 for 3 h at room temperature), anti VE cadherin (Abeam, Cambridge,Mass., 1:100 overnight at 4° C.) specific for EC (7), anti GFP (LifeTechnologies, 1:1000 overnight at 4° C.) antibodies. Fluorescent-labeledsecondary antibodies (1:1000) were incubated at room temperature for 1hour. Samples were then washed with PBS and mounted with SlowFade® Goldantifade reagent with DAPI (Life Technologies). Images were taken byusing a Nikon A1 scanning confocal microscope (Nikon Instruments,Melville, N.Y.) and acquired with NIS-Elements advanced researchsoftware. Images were optimized for contrast, without any furthermanipulation. Neointima/media ratios were calculated using acomputerized image analysis system (Image J), as previously described(4).

Vascular Reactivity.

After isolation from the rats, common carotid arteries were suspended inisolated tissue baths filled with 25 mL Krebs-Henseleit solution (inmMol/L: NaCl 118.3, KCl 4.7, CaCl₂ 2.5, MgSO4 1.2, KH₂PO₄ 1.2, NaHCO325, and glucose 5.6) continuously bubbled with a mixture of 5% CO₂ and95% O₂ (pH 7.38 to 7.42) at 37° C. as previously described (8).Endothelium-dependent vasorelaxation was assessed in vesselspre-constricted with phenylephrine (10⁻⁵ M) in response to acetylcholinefrom 10⁻⁹ to 10⁻⁶ M, freshly prepared on the day of experiment (5, 9).Concentrations are reported as the final molar value in the organ bath.Endothelium-independent vasodilatation was tested after mechanicalendothelium removal of the endothelial layer.

REFERENCES

-   1. Ciccarelli, M., et al. Endothelial alpha1-adrenoceptors regulate    neo-angiogenesis. Br J Pharmacol 153, 936-946 (2008).-   2. Santulli, G., et al. Evaluation of the anti-angiogenic properties    of the new selective alpha Vbeta3 integrin antagonist RGDechiHCit. J    Transl Med 9, 7 (2011).-   3. Totary-Jain, H., et al. Rapamycin resistance is linked to    defective regulation of Skp2. Cancer Res 72, 1836-1843 (2012).-   4. Iaccarino, G., Smithwick, L. A., Lefkowitz, R. J. & Koch, W. J.    Targeting Gbeta gamma signaling in arterial vascular smooth muscle    proliferation: a novel strategy to limit restenosis. Proc Natl Acad    Sci USA 96, 3945-3950 (1999).-   5. Iaccarino, G., et al. AKT participates in endothelial dysfunction    in hypertension. Circulation 109, 2587-2593 (2004).-   6. Yamaguchi, K., et al. Local persistent hypercoagulability after    sirolimus-eluting stent implantation in patients with stable angina.    Int J Cardiol 153, 272-276 (2011).-   7. Vestweber, D. VE-cadherin: the major endothelial adhesion    molecule controlling cellular junctions and blood vessel formation.    Arterioscler Thromb Vase Biol 28, 223-232 (2008).-   8. Santulli. G., et al. In vivo properties of the proangiogenic    peptide QK. J Transl Med 7, 41 (2009).-   9. Santulli, G., et al. CaMK4 Gene Deletion Induces Hypertension. J    Am Heart Assoc 1, e001081 (2012).

Example 2

Coronary artery disease is currently a leading cause of death worldwide.

Despite all the benefits of drug-eluting stents (DES), concerns havebeen raised over their long-term safety, with particular reference tostent thrombosis due to delayed endothelial cell (EC) coverage.Described herein is a method that exploits the endogenous miRNAs tospecifically inhibit vascular smooth muscle cell (VSMC) proliferation,the major cause of restenosis, without affecting reendothelialization.

By inserting four target sequences of the EC specific mir-126 into the3′UTR of p27 expressing adenoviruses (p27-126TS), VSMC proliferation andmigration was specifically inhibited, while EC were able to proliferate,migrate and form capillary-like networks. Balloon injured rat carotidartery infected with p27-126TS viruses exhibited complete inhibition ofrestenosis with complete re-endothelialization after two weeks (FIG.3B). Moreover, hypercoagulability assessed by measuring plasma levels ofD-dimer in the serum of rats treated with the p27-126TS was similar tothe non-injured control (FIG. 3D). Finally, the vasodilatative responseto acetylcholine of balloon injured carotid arteries treated withp27-126TS adenovirus were comparable to those of the control-noninjuredvessel (FIG. 3E). The data above can lead to a safer therapy than thenon-selective DES and can diminish the need for prolonged dualantiplatelet therapy following percutaneous coronary intervention.

Example 3

Antithrombotic and/or antiinflammatory genes will be incorporated intothe adenovirus expression vectors described in the examples above, orinto any other DNA delivery system, as described in the DetailedDescription. Such genes will be incorporated into the adenovirus vectorsunder the control of a second CMV promoter.

Example antithrombotic and/or antiinflammatory genes include thefollowing:

-   -   Ectonucleoside triphosphate diphosphohydrolase, ENTPD1. The        nucleic acid sequence of the gene encoding ENTPD1, including,        but not limited to, the nucleic acid sequence of the open        reading frame of the gene, is known in the art. The nucleic acid        sequence of the gene encoding human ENTPD1, including, but not        limited to, the nucleic acid sequence of the open reading frame        of the human gene, is known in the art. The amino acid sequences        of the ENTPD1 polypeptide and protein, including, but not        limited to, the amino acid sequences of the human ENTPD1        polypeptide and proteins, are known in the art. The GenBank        accession number of a polypeptide sequence of human ENTPD1 is        AAH47664.    -   Prostacyclin synthase, PTGIS. The nucleic acid sequence of the        gene encoding PTGIS, including, but not limited to, the nucleic        acid sequence of the open reading frame of the gene, is known in        the art. The nucleic acid sequence of the gene encoding human        PTGIS, including, but not limited to, the nucleic acid sequence        of the open reading frame of the human gene, is known in the        art. The amino acid sequences of the PTGIS polypeptide and        protein, including, but not limited to, the amino acid sequences        of the human PTGIS polypeptide and proteins, are known in the        art. The GenBank accession number of a polypeptide sequence of        human PTGIS is BAA11910.    -   Tissue factor pathway inhibitor, TFPI. The nucleic acid sequence        of the gene encoding TFPI, including, but not limited to, the        nucleic acid sequence of the open reading frame of the gene, is        known in the art. The nucleic acid sequence of the gene encoding        human TFPI, including, but not limited to, the nucleic acid        sequence of the open reading frame of the human gene, is known        in the art. The amino acid sequences of the TFPI polypeptide and        protein, including, but not limited to, the amino acid sequences        of the human TFPI polypeptide and proteins, are known in the        art. The GenBank accession number of the nucleic acid sequence        of human TFPI is AF021834.

What is claimed is:
 1. A nucleic acid vector comprising a gene ofinterest, and one or more target sequences for a microRNA within the 3′UTR region of the gene of interest.
 2. The nucleic acid vector of claim1, wherein the gene of interest is the p27 gene.
 3. The nucleic acidvector of claim 1, wherein the microRNA is miR-126.
 4. The nucleic acidvector of claim 3, wherein the one or more target sequences comprise SEQID NO:2.
 5. The nucleic acid vector of claim 1, wherein the vectorcomprises four target sequences for a microRNA.
 6. The nucleic acidvector of claim 5, wherein the target sequences for a microRNA areidentical.
 7. The nucleic acid vector of claim 6, wherein the targetsequences for a microRNA comprise SEQ ID NO:2.
 8. The nucleic acidvector of claim 1, wherein the vector comprises a second gene ofinterest.
 9. The nucleic acid vector of claim 8, wherein the vectorcomprises one or more target sequences for a microRNA within the 3′ UTRregion of the second gene of interest.
 10. The nucleic acid vector ofclaim 1, wherein the nucleic acid vector is a viral vector.
 11. Thenucleic acid vector of claim 10, wherein the viral vector is anadenoviral vector.
 12. The nucleic acid vector of claim 1, wherein thevector is delivered to a cell of interest.
 13. The nucleic acid vectorof claim 12, wherein the cell of interest is an endothelial cell.
 14. Anexpression system comprising a nucleic acid vector, wherein the nucleicacid vector comprises a gene of interest, and one or more targetsequences for a microRNA within the 3′ UTR region of the gene ofinterest.
 15. The expression system of claim 14, wherein the gene ofinterest is the p27 gene.
 16. The expression system of claim 14, whereinthe microRNA is miR-126.
 17. The expression system of claim 16, whereinthe one or more target sequences comprise SEQ ID NO:2.
 18. Theexpression system of claim 14, wherein the vector comprises four targetsequences for a microRNA.
 19. The expression system of claim 18, whereinthe target sequences for a microRNA are identical.
 20. The expressionsystem of claim 19, wherein the target sequences for a microRNA compriseSEQ ID NO:2.
 21. The expression system of claim 14, wherein the vectorcomprises a second gene of interest.
 22. The expression system of claim21, wherein the vector comprises one or more target sequences for amicroRNA within the 3′ UTR region of the second gene of interest. 23.The expression system of claim 14, wherein the nucleic acid vector is aviral vector.
 24. The expression system of claim 23, wherein the viralvector is an adenoviral vector.
 25. A cell comprising a nucleic acidvector, wherein the nucleic acid vector comprises a gene of interest,and one or more target sequences for a microRNA within the 3′ UTR regionof the gene of interest.
 26. The cell of claim 25, wherein the gene ofinterest is the p27 gene.
 27. The cell of claim 25, wherein the microRNAis miR-126.
 28. The cell of claim 27, wherein the one or more targetsequences comprise SEQ ID NO:2.
 29. The cell of claim 25, wherein thevector comprises four target sequences for a microRNA.
 30. The cell ofclaim 29, wherein the target sequences for a microRNA are identical. 31.The cell of claim 30, wherein the target sequences for a microRNAcomprise SEQ ID NO:2.
 32. The cell of claim 25, wherein the vectorcomprises a second gene of interest.
 33. The cell of claim 32, whereinthe vector comprises one or more target sequences for a microRNA withinthe 3′ UTR region of the second gene of interest.
 34. The cell of claim25, wherein the nucleic acid vector is a viral vector.
 35. The cell ofclaim 34, wherein the viral vector is an adenoviral vector.
 36. The cellof claim 25, wherein the cell is an endothelial cell.
 37. The cell ofclaim 25, wherein the cell is a vascular smooth muscle cell.
 38. Apharmaceutical composition comprising a nucleic acid vector, wherein thenucleic acid vector comprises a gene of interest, and one or more targetsequences for a microRNA within the 3′ UTR region of the gene ofinterest.
 39. The pharmaceutical composition of claim 38, wherein thegene of interest is the p27 gene.
 40. The pharmaceutical composition ofclaim 38, wherein the microRNA is miR-126.
 41. The pharmaceuticalcomposition of claim 40, wherein the one or more target sequencescomprise SEQ ID NO:2.
 42. The pharmaceutical composition of claim 41,wherein the vector comprises four target sequences for a microRNA. 43.The pharmaceutical composition of claim 42, wherein the target sequencesfor a microRNA are identical.
 44. The pharmaceutical composition ofclaim 43, wherein the target sequences for a microRNA comprise SEQ IDNO:2.
 45. The pharmaceutical composition of claim 38, wherein the vectorcomprises a second gene of interest.
 46. The pharmaceutical compositionof claim 45, wherein the vector comprises one or more target sequencesfor a microRNA within the 3′ UTR region of the second gene of interest.47. The pharmaceutical composition of claim 38, wherein the nucleic acidvector is a viral vector.
 48. The pharmaceutical composition of claim47, wherein the viral vector is an adenoviral vector.
 49. A method oftreating or preventing a cardiovascular disease in a subject in needthereof, the method comprising administering a nucleic acid vector tothe subject, wherein the nucleic acid vector comprises a gene ofinterest and one or more target sequences for a microRNA within the 3′UTR region of the gene of interest.
 50. The method of claim 49, whereinthe gene of interest is the p27 gene.
 51. The method of claim 49,wherein the microRNA is miR-126.
 52. The method of claim 51, wherein theone or more target sequences comprise SEQ ID NO:2.
 53. The method ofclaim 52, wherein the vector comprises four target sequences for amicroRNA.
 54. The method of claim 53, wherein the target sequences for amicroRNA are identical.
 55. The method of claim 54, wherein the targetsequences for a microRNA comprise SEQ ID NO:2.
 56. The method of claim49, wherein the vector comprises a second gene of interest.
 57. Themethod of claim 56, wherein the vector comprises one or more targetsequences for a microRNA within the 3′ UTR region of the second gene ofinterest.
 58. The method of claim 49, wherein the nucleic acid vector isa viral vector.
 59. The method of claim 58, wherein the viral vector isan adenoviral vector.
 60. The method of claim 49, wherein the nucleicacid vector is delivered into a cell of the subject.
 61. The method ofclaim 60, wherein the cell expresses the microRNA.
 62. The method ofclaim 60, wherein the gene of interest is not expressed in the cell. 63.The method of claim 60, wherein the cell is an endothelial cell.
 64. Themethod of claim 60, wherein the cell is a vascular smooth muscle cell.65. The method of claim 60, wherein the cardiovascular disease iscoronary artery disease.